Anal. Chem. 1981, 53, 992-997
992
Automated Determination of Ammonia with a Potentiometric Gas Sensor and Flowing Internal Electrolyte Yvonne M. Fraticelli and M. E. Meyerhoff" Depaament of Chemistry, The University of Michigan, Ann Arbor, Michisan 48 709
An electrode-based automated method for rapid determination of ammonla nitrogen In the 10-8-10-3 mol/L range Is described. The system deslgned utilizes a novel tubular flowthrough poly( vlnyl chloride)-nonactln membrane electrode to sense ammonium ions formed In a recipient Internal buffer stream as the sample passe8 through a gas dialysis chamber. Peak potentials observed are logarlthmlcally related to sample ammonla-N concentrations. At least 30 samples/h can be determined with excellent preclslon (f1 mV standard deviatlon) for samples I mol/L. Thorough investigallons regardlng the optimization of system varlables such as flow rates, diluent reagents, segmentation, etc. are described. Prellmlnary studies are presented which demonstrate the immediate appllcatlon of thls System for the determination of ammonla in serum, plasma, and whole blood samples.
Potentiometric ion-selective membrane electrodes have become widely used as detectors in continuous flow automated systems for the determination of discrete ions in complex biological, environmental, and industrial samples (1-5). Similar efforts to utilize pH glass membrane based gas-sensing electrodes for such purposes have been less successful due to the diffusion-controlled slow response of these sensors, pafticularly upon return to base line potentials. In practice, this results in low sample throughput capabilities and much poorer detection limits. We recently developed a new polymer membrane electrode-based ammonia-selective sepsor (6) and further demonstrated its advantageous detection limits vs. traditional sensors by developing a manual blood ammonia method (7). We now describe the incorporation of this gassensing concept into an autoanalyzer system for the rapid determination of ammonia nitrogen (ammonia-N) a t trace levels. (Ammonia-N refers to total ammonia nitrogen present, that is, the sum of the ammonium ions and free ammonia gas in solution). Previous workers have incorporated commercially available membrane type and air gap type ammonia gas sensors into segmented autoanalyzer or flow injection systems by utilizing appropriate flow-through caps (8-12). While these reports suggest that between 20 and 60 samples/h can be assayed, such throughput can only be obtained for samples with relatively high ammonia-N levels (>lo4 mol/L). Indeed, in most instances, a significant quantity of ammonia has been added to the wash s o l u t i h to keep a steady reproduoible base line potential. Such problems arise due to the slaw rate of ammonia diffusion between the static thin film of electrolyte within the sensor and the flowing sample stream. Recently, the use of a flowing internal electrolyte concept in conjunction with a gas dialysis unit has been suggested as a general remedy for such problems (13,14). Durst (13)used this approach to accurately detect hydrogen cyanide a t low levels using a silver ion selective membrane electrode. Technicon Instruments now incorporates this gas dialysis concept to detect carbon dioxide in physiological samples (14). Surprisingly, no report of a similar system for ammonia measurements has been made to date.
The development of a simple, accurate, sensitive, and selective automated ammonia assay would find wide applications in numerous disciplines. Perhaps the most urgent current need for such a system lies in the area of clinical chemistry. The clinical assay of ammonia-N in blood samples has found increasing importance in the diagnosis of sever4 disease states including hepatic coma, Eck's fistula, and Reye's Syndrome (15-18). Current automated spectrophotometric ammonia methods (e.g., Nessler's etc.) are not sensitive enough to detect normal blood ammonia levels which typically fall between 10 and 80 pmol/L (15,17).An enzymatic procedure based on the catalytic action of glutamate dehydrogenase is the only automated system widely used to date (DuPont ACA (18)). The method lacks precision at low plasma ammonia levels and suffers interferences from sample turbidity. In addition, it requires expensive commercial equipment and costly biochemical reagents (e.g., enzyme and cofactor). In this report we describe the development of a new automated electrode-based ammonia-selective assay which utilizes a flowing internal electrolyte technique. Measurements are based on the electrochemical detection of ammonium ions formed in a recipient stream of internal buffer as the sample flows through a gas dialysis assembly. A novel tubular flowthrough PVC-nonactin membrane electrode is used to sense the ammonium ions formed in the recipient stream. Peak potentials observed are logarithmically related to ammonia-N concentrations. Emphasis has been placed on optimizing this new system for low-level blood ammonia measurements although the system described could readily be modified for ammonia determinations in most other samples. Detection limits using a sample stream pH of 8.5 are below mol/L ammonia-N. At least 30 samples/h can be readily assayed with high precision in the normal blood ammonia-N region. Preliminary studies are presented which demonstrate the application of this new systep for the direct determination of ammonia-N in whole blood as well as serum and plasma samples.
EXPERIMENTAL SECTION Apparatus. A schematic diagram of the automated ammonia-N system developed is shown in Figure 1. The sampler and proportioning pump were part of a Technicon (Tarrytown,NY) Autoanalyzer I system. Two matched multichannel AA-II,l2-in. dialysis plates fitted with a polytetrafluoroethylene (PTFE) gas-permeable membrane were used as the gas dialysis unit. All potentiometricmeasurements were made with an Altex Select-Ion 2000 ion analyzer (ScientificProducts, Romulw, MI) and recorded on a Houston Instruments (Austin,TX) Omni-Scribestrip chart recorder. Measurements were made at room temperature. Reagents. All chemicals used were reagent grade. Standard solutions and working buffers were prepared with distilleddeionized water. Nonactin was obtained from Sigma Chemical Co. (St. Louis, MO). Poly(viny1chloride) (PVC), chromatographic grade, was obtained from Polysciences, Inc. (Warrington, PA). Dibutyl sebecate was a product of Eastman Kodak (Rochester, NY). Working buffers studied for both the flowing internal electrolyte stream and the sample-diluent stream included the following: tris(hydroxymethy1)atninomethane-hydrogen chloride (Tris-HCl), pH 7.5,0.01 mol/L; Tris-HC1, pH 8.5,0.02,0.015, and 0.05 mol/L;
0003-2700/81/0353-0992$01.25/00 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 7, JUNE 1981 S93
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Schematic diagram of automated ammonia-N assay system. Key: (REC) recorder; (pH) pKmV meter; (r) saturated calomel reference electrode; (e) electrolyte solution; (sb) salt bridge; (fce)flow cell polymer eleCtrode unit; (g)'electrical ground; (m) gag-permeable membrane; (dc) dialysis chamber; (ps) pulse suppressor; (d) debubbler; (mc) seven-turn mixing coil; (w) waste; (HO) cactus connector. Flgure 1.
Tris-HC1, pH 9.2, 0.015 mol/L; 1,3-bis[tris(hydroxymethyl)methylamino]propane-hydrogen chloride (Bis-Tris-HCl),pH 6.5, 0.01 mol/L; NaOH, 0.01,0.015, and 0.0015 mol/L (buffer concentrations refer to total ionic strength). Lyophilized normal control serum was obtained from Fisher Scientific Co. (Livonia, MI) (Sera Chem lot no. 2905-09) and reconstituted with distilled-deionized water. Dowex 50W-X8 (100-200 mesh) from J. T. Baker Chemical Co. (Glen Ellyn, IL) was converted to the Na+ form and dried in an oven at 100 "C prior to use. Construction of Tubular Flow-Through Ammonium-Selective Polymer Membrane Electrode Unit. In the initial phase of this study, various flow-throughelectrodes, incorporating our earlier PVC-nonactin ammonium-selective membrane materials (6, 7), were designed and evaluated for use in our autoanalyzer system. The design which proved the most useful in terms of overall performance and simplicity of preparation is shown schematically in Figure 2. This design is a modification of a British patent (19)which describes the fabrication of a tubular flow cell assembly for the determination of potassium using a PVC-Valinomycin membrane. The compositionof the PVC-nonactin membrane was modified slightly from that reported in our original static ammonia electrode studies (6). Twenty milligrams of PVC was used rather than 65 mg to prepare the casting solution containing nonactin, plasticizer (dibutyl sebecate), and THF (tetrahydrofuran). Subsequent studies have shown that for this particular tubular design, the ion-selective membrane had improved detection limits and a more Nernstian response to ammonium when the PVC content was reduced. This may be due to the increased fluidity of the resulting membrane. The final electrode unit was prepared in the following manner: a hole of approximately 4 mm diameter was cut out of the side of a 40 mm long PVC tube (of 0.89 mm). A disposiable 20 gauge syringe needle (1.5 in. long) with its tip rounded off, was used as a mandrel and inserted within the tube beneath the cut opening. This was done to ensure a smooth cyclindrical surface inside the tube as the membrane formed over the hole. The PVC-nonactin casting mixture was then deposited over the opening in a dropwise manger. The drops were big enough to cover the entire opening without overflowing. Each drop was left to dry before depositing the next. This was repeated 5 times. The membrane formed as the volatile solvent, THF, evaporated. The THF also dissolved the PVC tube at the cut edges, which resulted in the chemical bonding of the membrane to the tube. The body of the electrode consisted of a plastic pipet tip which was heat sealed a t the narrow end. Two holes of the same diameter as the PVC tube were drilled through the plastic body. The tube was inserted through these holes with the ion-selective membrane facing down and sealed in place with a PVC-cyclohexanone paste to prevent leakage of the internal reference electrolyte (0.01 mol/L "&I). Downstream from this electrode unit, one end of a lithium acetate salt bridge was inserted into the flowing solution and the
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Flgure 2. Schematic diagram of tubular ammonium selective potymer membrane electrode flow unit: (A) coaxial cable, (B) plastic pipet tips, (C) AglAgCl electrode, (D) lo-* ml/L NH,CI, (E) PVC tubing (0.89 mm.), (F) PVC-oonactin ammonium selective membrane.
electrochemical circuit was completed when the other end of the salt bridge and a saturated calomel electrode were immersed into a beaker containing buffer (see Figure 1). Lithium acetate was specificially used as the salt bridge electrolyte (over sodium and potassium salts) to avoid possible leakage of interfering cations into the recipient buffer stream. The PVC-nonactin electrode has only slight response to L P (6). Evaluation of Automated Ammonia System. Figure 1 schematically illustrates a typical flow arrangement used to evaluate our new gas-sensing system when using aqueous NH4Cl standards. As shown, the sample is diluted with an appropriate diluent in a slightly greater than 1:2 mixing ratio. As detailed later (Results and Discussion), numerous variations in flow rates, air segmentation intervals, dilution ratios, diluent buffers, etc. were tried throughout the courge of tgis study. Since electrode response varied significantiywith tihe, whenever pwible, specific comparative studies were carried out on a single day so that electrode behavior would be constant *oughoqt %e experiments. In general, for evaluation of the system, standard aqueous mol/L were solutions of NH4Cl ranging from 1 )4 10" to 1 X placed in the sample tray. At the beginning of the day, the pump was turned on allowing the reagents to flow through the system.
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After a few minutes, a steady base line potential was observed and sampling commenced. Peak potentials for each sample were determined from the difference between the initial known base line value and the calibrated recorder span. Whenever a new reagent stream was introduced to the system, a new base line potential was first observed prior to any sampling. Procedure for Blood Ammonia Determinations. A calibration plot of aqueous standards ranging between 10 and 100 pmol/L NH,CI was recorded prior to sampling the specimens. The sampling rate used was 30 samples/h with a sample to wash ratio of 1:2. The diluent stream was a 0.05 mol/L Tris-HC1buffer, pH 8.5. The blood samples were diluted 1:4 within the system with this buffer stream by using appropriate flow-rate pump tubing. The resultant sample stream, with a total flow rate of 6.10 mL/min (sample + diluent + air), was debubbled prior to flowing through the dialysis unit and further pulled through at a rate of 3.90 mL/min. The flow rate of the internal recipient buffer stream (0.01 mol/L Tris-HC1, pH 7.5) was 2.00 mL/min. Serum samples were obtained from the University of Michigan Hospital‘s Stat Laboratory (Ann Arbor, MI). Samples were stored on ice until assayed (usually 2-3 h after being drawn). Whole blood samples were volunteered by healthy members of our research group. These blood samples were drawn into heparinized evacuated tubes at the University of Michigan’s Health Services and immediately placed on ice. Portions of the whole blood samples were centrifuged to obtain the corresponding plasma samples. All of these samples were assayed within 30 min. Unknown blood ammonia values were determined from the prior aqueous calibration curves using a linear “least-squares fit” of the data. Procedure for Serum Recovery Studies. Since all commercial control serum tested had extremely high levels of ammonia-N, it was necessary to reduce this ammonia-N level to normal values before useful analytical recovery experiments could be undertaken. The following procedure was used for such purposes: Approximately 1g of Dowex 50W-X8 cation exchanger in Na+ form was added to 10 mL of reconstituted control serum and mixed throughly. The resin-serum slurry was separated by centrifugation. This procedure was repeated twice. This method does not remove all the NH3-N in the sample but does reduce the levels significantly. To each of 2 mL of final supernatant, microliter quantities of and mol/L NH4C1standards were added, mixed, and then assayed.
RESULTS AND DISCUSSION The automated ammonia gas sensing system described in this report utilizes the same chemical detection principles as our earlier static-type ammonia sensor (6). In that work, an ammonium selective polymer membrane electrode was used to detect ammonium ions formed, from diffusing ammonia gas, in a thin film of buffer at the tip of the sensor. By substitution of a polymer electrode for the more traditional pH glass internal probe, a significant improvement in detection limits was achieved, owing to a buffer trep effect (6,20). In developing this new automated ammonia assay, we felt these same improvements could be attained if we similarly utilized an ammonium-selective electrode as the ultimate detector. Moreover, due to its simple design, ruggedness, and extreme low cost, we felt the polymer ammonium electrode would be much simpler to incorporate into a flowing system. As seen in Figure 1, the system designed allows a sample to mix with a diluent solution which has a pH favoring the formation of free ammonia gas from the total ammonia-N in the sample. The sample-diluent stream then passes through a gas dialysis chamber where free gas diffuses across a gas-permeable membrane into a recipient buffer stream of lower pH, thus forming ammonium ions. The quantity and rate of gas transfer are aided by the pH difference between the two streams. Figure 3 illustrates an expanded view of the gas dialysis unit and the overall chemical processes on which this system is based. The amount of ammonium ions formed in the recipient (top) stream is potentiometrically sensed as that portion of the stream flows through the ammonium selective polymer membrane electrode (Figure 2). In such an arrangement, when no NH, is present in the sample stream, a completely fresh portion of the internal buffer stream
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