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ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978

Automated Separation and Conductimetric Determination of Ammonia and Dissolved Carbon Dioxide
Robert M. Carlson
Department of Pomology, University of California, Davis, California 956 76

A continuous flow instrument for automated determination of ammonia or dissolved carbon dioxide has been developed. The sample solution stream is mixed with sodium hydroxide for ammonia determination of perchloric acid for carbon dioxide determination. The ammonia or carbon dioxide diffuses from the sample stream through silicone rubber hollow fibers into a stream of deionized water. As the water stream emerges from the hollow fibers, it passes through an electrical conductivity cell. The conductivity response is related to ammonium or carbon dioxide in the sample. The average relative standard deviation for total nitrogen in 39 leaf samples digested by the Kjeldahl procedure was 0.66%. Agreement between the new method and distillation-titration of Kjeldahl digests was very good. Recovery of nitrogen for the Bureau of Standards orchard leaf reference sample was within one standard deviation of the certified value.

Various procedures for the determination of ammonia by electrical conductivity measurements have been reported. Hendricks et al. ( I ) described a vacuum distillation procedure in which the ammonia was collected in boric or sulfuric acid and determined by the change in conductivity of the acid. In a similar procedure, Appleton (2)distilled ammonia into boric acid, diluted the solution to known volume, and determined ammonia from conductivity changes. Shaw and Staddon ( 3 ) used a diffusion cell to transfer ammonia from the sample to sulfuric acid for its subsequent determination by conductivity. Fried1 ( 4 ) determined ammonia from the rate of change of conductivity of a small volume of sulfuric acid as it absorbed ammonia from a sample in a diffusion cell. Separation of ammonia by distillation ( 5 ) and gas diffusion (6) have been employed in automated colorimetric methods. Diffusion through plastic tubing has been reported as a technique for separating gaseous components of samples. Kollig et al. (7) used silicone rubber tubing in a device for sampling waters for dissolved oxygen, nitrogen, and carbon dioxide determinations. Scarano and Calcagno (8) determined dissolved carbon dioxide from p H changes in a bicarbonate solution flowing through a Teflon tube immersed in the sample. Westover et al. (9) descrihed a sampling device for mass spectrometry that was based on gas permeation through silicone rubber hollow fibers. The work reported here was carried out to develop a system for automated ammonia determinations in biological samples after Kjeldahl digestion. The method is based on the transfer of ammonia by diffusion through silicone rubber hollow fibers into a flpwing stream of water, followed by detection by electrical conductivity. The apparatus was also found to be useful for ammonia determination in water samples and for dissolved carbon dioxide determinations.

EXPERIMENTAL Apparatus. The gas permeation tube (Figure 1)was prepared by attaching an */*-in.i.d. polypropylene Y connector t o each end of a 50-cm length of 0.062-in. i.d. small bore polyethylene tubing
0003-2700/78/0350-1528$01 .OO/O

with short sections of silicone rubber tubing (1,/,6-in. i.d., 3/ls-in. 0.d.). Twelve strands of Dow Corning silicone rubber hollow fibers (obtained from Bio-Rad Laboratories, Richmond, Calif.) were placed in the tube and potted into one branch of the Y connector on each end with Dow Corning silicone rubber sealer. The electrical conductivity cell is shown schematically in Figure 2. The well formed with the various sizes of vinyl tubing was filled with styrene casting resin to hold the electrodes of the cell rigidly in place. This construction permits placement of the conductivity cell very close to the otitlet of the hollow fiber bundle. Figure 3 is a block diagram of the complete apparatus. Components used to assemble the apparatus are as follows: Arthur H. Thomas Co., Little Dipper Automatic Sampler, equipped with a timer adjustable from 0.2 to 5 min; Gilson Model HP-4, 4channel peristaltic pump; Thermonix Model 1480 Constant Temperature Circulator; Markson Science, Inc. Model 4405 Conductivity Analyzer; and Linear Instruments Corp. Model 254 Strip Chart Recorder. The sample and NaOH (or HC10J streams are mixed in a micromixing device constructed as described by Gugger and Mozersky (10). Air is injected into the mixed sample stream just as it exits the micromixer. The stream then enters the thermostated water bath and passes through a 50-cm coil of 2-mn id., 1-mm wall glass tubing to bring the sample to bath temperature. It then passes through the tube containing the hollow fibers and is discharged t,o waste. Dionized distilled water is pumped through a small column of mixed bed resin for further polishing. This small column was constructed from the polypropylene barrel of a 6-mL disposible syringe. The resin is supported in the column by disks of porous polyethylene. As the water stream leaves this column, it passes through a 25-mm Swinnex filter holder with a Millipore type AA membrane to remove any particulate matter that might clog the hollow fibers. The water stream then enters the thermostated bath, passes through the degassing unit, another small, mixed bed deionizing column (constructed as above but with a 1-mL syringe barrel), through the hollow fibers, the conductiviy cell, and is discharged to waste. The degassing unit consists of a silicone rubber hollow fiber minitube (Bio-Rad Laboratories) with the outer chamber connected to a house vacuum system through a 1-L vacuum flask ballast and a check valve. Inclusion of the degasser prevents air bubble formation in the hollow fibers and the conductivity cell when the bath is operated at elevated temperature. The sample, reagent, and water transmission lines were cut from 0.042-in. i.d. polyethylene tubing. Connections were made with silicone rubber or vinyl tubing. Reagents. All reagents were prepared from analytical reagent grade chemicals. Samples were mixed with 1.5 M NaOH cotaining 6 g Na2EDTA.2H20/L. The EDTA was added to prevent precipitation of polyvalent metal hydroxides and carbonates. Carbon dioxide determinations were carried out by mixing the sample with 1 M HC104. Operating Procedure. The start-up procedure consisted of pumping water through the reagent and sample lines while the temperature bath stabilized (10 to 15 min.). These lines were also flushed with water for 5 min at instrument shutdown. When not in use, the degassing unit was disconnected from the vacuum line to prevent loss of water from the hollow fibers. Kjeldahl Digestion. Plant tissue samples, 250-mg aliquots, were digested in calibrated 75-mL digestion tubes in a block digestor. The digestion mixture was 5 mL HZSO,, 3.5 g KzS04, and 40 mg CuS04. One selenized boiling chip was added to each tube. The samples were heated to 370 "C and held at this
C
1978 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978

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Lv
I
"

FIBERS POTTED WITH SILICONE RUBBER

TUBING

r(P 0 LY PR O P Y L E NE Y
"

C O N N ECTOR

Flgure 1. Schematic diagram of silicone rubber hollow fiber permeation unit

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Figure 4. Typical recorder trace for standards and Kjeldahl nitrogen samples. Standards indicated by millimolar concentration of ammonium, samples indicated by number

Figure 2. Schematic diagram of electrical conductivity cell. A, B, C, D are vinyl tubing: (A) X 1 / 4 , (C) i / 4 X 3/8, (D) 3/a X ' I 8 ,(B) X '/* inches (i.d. X o.d.), (E) 6-cm section of 14 gauge stainless steel tubing, (F) leads to conductivity meter, (G) 0.8-mm diameter stainless steel wire

DElONlZlYO COLUMN

VACUUM

Flgure 3. Block diagram of complete instrument

temperature for 2 h. After cooling, they were diluted to 7 5 mL and mixed.

RESULTS AND DISCUSSION

Even though the surface area of the fiber bundle is large (estimated to be 47 cm2 for a 50-cm long unit containing 12 fibers), the pumping rate required to allow the water passing through the fibers to reach equilibrium with the sample is too slow for rapid processing of samples. The system depends on a steady-state transfer of gas rather than equilibration of the water stream with the sample stream. The diffusion of gas through the fiber walls is temperature-dependent as is the electrical conductivity of the resulting ammonium hydroxide or carbonic acid solutions. The overall temperature de-

pendence was determined by noting the response to an ammonium standard at various temperatures between 25 and 50 "C. The response increased by approximately 4% per degree. Hence, a bath that w i l l maintain temperature constant to i 0.01 "C provides adequate control of this variable. An estimate of the extent of gas transfer was obtained by collecting the water passing through the fibers while a M NH4Cl was pumped through the unit a t 4.98 mL/min mixed with 1.5 M NaOH a t 1.17 mL/min. The flow rate of water through the fibers was 0.98 mL/min and the bath temperature was 35 "C. The water from the fibers was collected in a small volume of 1 M HC10, to prevent volatilization losses. This solution was then run through the instrument as a sample with appropriate standards to determine the ammonium concentration, which was found to be 2.6% of the ammonium concentration in the NH4Cl/NaOH stream. For adaptation to samples with very low levels of ammonium, the extent of gas transfer could be increased by employing a unit with more and/or longer fibers and by operating a t higher temperatures. Decreasing the pumping rate of water through the fibers will also increase the concentration of ammonia in the solution reaching the conductivity cell. In addition to maintaining constant temperature, the pumping rates must be constant (especially that of the water passing through the fibers) to obtain stable response. The response time depends on the rate a t which sample and reagent are pumped over the fibers and on the rate water is pumped through the fibers. Typical sampling time used in this work was 90 s with a n additional 10 s required for the sampler to raise the probe and advance to the next sample. Air is aspirated between samples. This provides a convenient spike in the recorder trace that separates samples of similar composition. Figure 4 shows a typical recorder trace. The effect of the total concentration of salts in the sample stream is an additional variable that requires attention. The

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ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978

Table I. Comparison of Instrumental Nitrogen Determination with Distillation-Titration for Digested Leaf Samples N, 9 6 distillationsample instrumental titration apple pear peach almond mixed species prune prune prune prune prune
2.21 2.27 2.55 1.62 2.17 2.70 2.94 2.25 2.11 2.33 2.24 2.26 2.55 1.62 2.14 2.69 2.93 2.29 2.10 2.34

partial pressure of ammonia or carbon dioxide will depend on the total salt load. For water samples, the total concentration will be dominated by the NaOH or HC104 mixed with the sample, so sample to sample variation will be small. For digested samples, the sodium sulfate formed in the neutralization of sulfuric acid and the potassium sulfate in the digestion mixture will be major components of the salt load. Standards for these determinations should contain sulfuric acid and potassium sulfate a t concentrations similar to those of the samples. The amount of potassium sulfate required can be calculated but some sulfuric acid is consumed in the digestion so the acid concentration in a few samples should be determined to establish the amount of acid to be added to the standards. As the amount of acid consumed in the digestion will vary somewhat, the effect of variable acid concentration was determined. Ammonium chloride standards were prepared with 30, 60, and 90 mL sulfuric acid/L. Instrument response decreased 2.5% as acid concentration increased from 30 to 90 mL/L. The acid concentration for digested samples ranged from 50.5 to 55.5 mL/L. The effect of this variation is insignificant. Standards were prepared to contain the average acid concentration found in the samples to avoid systematic errors. Most of the precision and accuracy determinations were done with plant tissue samples that had been carried through the Kjeldahl digestion procedure. Precision was determined by running 39 digested plant leaf samples through the instrument in a different random order on each of 5 different days. These determinations were made on the same digested solutions in order to eliminate variability in the weighing and digestion of the samples. The average relative standard deviation for the 39 samples was 0.66%. The relative standard deviation exceeded 1% for only 2 of the samples. The precision of the instrument is quite good. Accuracy was determined by comparing the nitrogen found in the National Bureau of Standards Reference Material 1571 (Orchard Leaves) with the certified value and by comparing the nitrogen found with the instrument with that found by distillation and titration for ten samples of deciduous fruit tree leaves. The mean nitrogen concentration and standard deviation found for the Bureau of Standards sample was 2.72 f 0.02% (11 determinations). The certified value of this sample is 2.76 f 0.05%. The results comparing instrumental with distillation-titration methods are tabulated in Table I. The determinations by the two methods were carried out on the same digested samples to eliminate variability in the weighing and digestion of the samples. There is no significant difference between the methods. The usefulness of the apparatus for determination of ammonium in water samples depends on its response to low concentration of ammonium. The lower limit of response is determined by the changes in electrical conductivity as

Flgure 5. Calculated specific conductivity vs. ammonia concentration

Table 11. Comparison of Instrumental Determination of Carbonate plus Bicarbonate with Results from Titration C0,'- + HCO,-, mequiv/L sample instrumental titration drain water 1 6.69 6.8
2 3 4 well water 1 2

5.65 4.56 6.35 7.50 7.91

5.4 4.8 6.1 7.2 7.6

ammonia dissolves in water. The conductivity is a function of the concentrations of ammonium, hydroxide, and hydrogen ions. Addition of ammonia decreases hydrogen and increases ammonium and hydroxide ion concentrations. Hydrogen is much more mobile than ammonium ion so the change in conductivity with the initial addition of ammonia is very small. This can be shown by calculating the specific conductivity from the limiting ionic mobilities of the three ions and the ammonia-water equilibrium. Figure 5 shows the calculated conductivity as a function of the square root of ammonia concentrations at 25 "C. The limiting ionic mobilities used were: H', 350; OH-, 198; and NH4+,73.4. The dissociation constant for ammonium hydroxide was taken as 1.77 X The figure shows the lag in response with the initial addition of ammonia. Calibration curves obtained with dilute ammonium standards were consistent with the calculated response. When the sample, reagent, and water pumping rates were 4.98, 1.17, and 0.96 mL/min, the useful lower limit was about 10-5M NH4+in the sample. This is adequate for most routine water analysis. Conversion of the apparatus to determine total dissolved carbon dioxide is accomplished by switching the reagent line from sodium hydroxide to an appropriate acid. Perchloric acid was used in this study. Sulfuric acid should work equally well. Suitability for dissolved carbon dioxide determinations was tested by comparing results for six drain water and well water samples with carbonate plus bicarbonate concentrations determined by titration. Concentrations of carbonate and bicarbonate were computed from total r!issolved carbon dioxide determined by the instrument, the dissociation constants of carbonic acid, and the pH of the water samples. Standards were prepared from sodium bicarbonate. The results are tabulated in Table 1 1 . The agreement between the methods

ANALYTICAL CHEMISTRY, VOL. 50, NO. 1'1, SEPTEMBER 1978

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is adequate for routine water analysis. Volatile acids or bases that would diffuse through the silicone fibers will interfere with carbon dioxide or ammonium determinations. Nitrate, chloride, and sulfate salts produced no response when the instrument w a s set up for carbon dioxide determinations. Acetate did produce a response and would interfere. Volatile amines are the most significant interferences in ammonium determinations. A test of interference from methylamine and dimethylamine showed a greater instrument response to these compounds than to ammonium solutions of the same concentration. Sample color and turbidity have no effect on performance of the instrument. The prototype instrument has required little maintenance beyond periodic replacement of the pump tubes. The hollow fiber unit shows no signs of deterioration after 24 months of use. The electrical conductivity detection system requires much less electrical shielding than potentiometric devices. There is no detectable drift in the baseline after a 5- to 10-min warm-up period. The maximum drift in response to standards observed was about 1%over a 3-h period. Segmenting the sample stream with air shortens the response time but the instrument can be operated without air injection if slightly longer sampling times are used. There is no need for debubbling the sample stream. The instrument is readily adapted to different concentration ranges by changing the sensitivity of the conductivity meter or by changing the size of the sample and reagent pump tubes. Preliminary observations indicate additional applications of the instrument. Adaptation t o continuous sampling should present no problem. I t responds to atmospheric carbon dioxide when air is pumped in place of sample and reagent so it may have use in gas analysis. It may be possible to use it

for nitrate determinations by passing the sample stream over a bed of Devarda alloy to convert nitrate to ammonia in a manner similar to that described by Mertens et al. (11). The method described here has some advantages over methods currently in use for ammonia determination. Compared with the distillation-titration procedure, it is faster, easier to automate, and can be used with smaller samples. The reagents are simpler than those used in colorimetric procedures and the method will tolerate colored or turbid samples which could not be run colorimetrically without pretreatment. Samples with high concentrations of solutes can be analyzed if standards contain approximately the same concentration of solutes. The gas sensing ammonia electrode will not tolerate high solute concentrations. Electronic drift is much less that that encountered with the ammonia electrode. The advantages of this new method should make it an attractive alternative to the methods currently in use.
LITERATURE CITED
(1) R. H.Herdricks,M. D.Thomas, M. Stout, and B. Tolman, I&. Eng. Chem., Anal. Ed., 5, 23-26 (1942). (2) L. Appieton, Chemist-Analyst,42, 4-7 (1953). (3) J. Shaw and B . W. Staddon, J . Exp. Biol., 35, 85-95 (1958) (4) F. E. Friedl, Anal. Biochem., 48, 300-306 (1972). (5) J. Keay, and P. M. A. Menage, Analyst(London), 94, 895-899 (1969). (6) Technicon Corporation, Tarrytown, N.Y., Industrial Method No. 330-74A. (7) H.P. Kollig, J. W. Falco. and F. E. Stancil. Jr.. Environ. Sci. Techno/., 9, 957-960 (1975). (8) E. Scarano and C. Calcagno, Anal. Chem., 47, 1055-1065 (1975). (9) L. B. Westover, J. C. Tou, and J. H. Mark, Anal. Chem.. 46, 568-571 (1974). (10) R. E. Gugger and S. M. Mozersky, Anal. Chem., 45, 1575-1576 (1973). (1 1) J. M e n s , P. Vandenwinkel, and D.L. Massart, AM/. Chem.,47, 522-526 (1975).

RECEIVED for review April 11,1798. Accepted June 23,1978.

Membrane Electrode Measurement of Lysozyme Enzyme Using Living Bacterial Cells

Paul D'Orazio, M. E. Meyerhoff, and G. A. Rechnitz'
Department of Chemistry, University of Delaware, Newark, Delaware

1971 1

Llvlng bacterial cells of the strain Micrococcus lysodeiktlcus are used as substrate for the determination of lysozyme actlvlty. The cells are loaded with a marker ion which Is released through the action of lysozyme upon the cell wall. The rate of ion release is monitored with a highly selective membrane electrode and is readily related to the concentration of enzyme present. The proposed method has excellent sensitlvlty and offers advantages of precision and convenience over previous turbidimetric methods.

Recently it has been shown that living bacterial cells can be used in conjunction with gas sensing electrodes to form bioselective sensors (1-3). So used, these cells are effectively serving as biocatalysts a t or near the electrode surface. Similarly, other vesicles such as sheep red blood cell ghosts loaded with electroactive marker, have been employed as analytical reagents for the determination of complement and antibodies by coupling the resultant lysing action to an appropriate ion selective electrode ( 4 ) . We now have extended

this approach by loading living bacterial cells with marker and using these cells as a reagent for the measurement of an antimicrobial enzyme such as lysozyme. Lysozyme (EC 3.2.1.17) catalyzes the hydrolysis of cell walls of several Gram-positive bacteria, with maximum activity toward Micrococcus lysodeikticus. The enzyme acts upon mucopolymers of these walls by breaking p-( 1-4)-glycosidic linkages between alternating N-acetylmuramic acid and N-acetylglucosamine residues ( 5 ) . Action of the enzyme in an osmotically protective medium results in formation of protoplasts which are intact living cells having a cell membrane but no cell wall (6). However, enzyme action in a hypotonic medium results in osmotic rupture of the membrane and release of the cytoplasmic material. Assays of lysozyme in clinical and research laboratories are most often carried out by a turbidimetric method, where decreases in absorbance of a M . lysodeihticus cell suspension are measured with time at 450 or 540 nm (7). The method suffers from a lack of precision and other problems arise from turbidity in the unknown sample. More recently immunochemical techniques have been suggested but results, thus far,

0003-2700/78/0350-1531$01.00/00 1978 American Chemical Society