Response of CaIcium-Selective and Divalent Cation-Selective Electrodes to Cyclohexylammonium Ion H. Bruce Collier Department of Pathology, University of Alberta, Edmonton 7, Alberta, Canada
IN THE COURSE of potentiometric measurements of binding of CaZ+ and Mg2+ by 2,3-diphosphoglycerate (DPG), it was observed, quite unexpectedly, that solutions of the pentacyclohexylammonium salt of DPG (Boehringer Mannheim Corp., New York, N. Y . ) increased the potentials of the electrodes. Potentiometric measurements were made with the Orion Model 92-20 calcium-selective and Model 92-32 divalent cation-selective electrodes against the Model 90-01 reference electrode at 25” in a buffered medium containing 50mM triethanolamine, 67mM KCl, and 13mM NaC1, adjusted to pH 7.5. Cyclohexylamine (Eastman Kodak No. 2496) was neutralized with HC1 to pH 7.5 and was titrated into the buffered medium. The responses of the two electrodes are given in Table I. At the lower concentrations the potential increments are almost Nernstian for a univalent cation (59 mV per decade). The cyclohexylammonium (CHA) cation was removed from a 0.1M solution of the DPG salt by repeated batchwise treatment with BioRad AG 50W X 8, H+ form (Calbiochem, Los Angeles, Calif.), and the resultant solution of the DPG free acid was neutralized with KOH. This solution had no effect on the potentials of the electrodes when added to the buffered medium. The cations of choline, triethanolamine, and tris(hydroxy-
Table I. Response of Electrodes to Cyclohexylammonium Cation Electrode potential, mV CHA concn, mM Model 92-20 Model 92-32 1 10
100 1000
- 66
- 32 +18 +75
- 12 +46
+120
+86
methy1)aminomethane (Tris, THAM) had no effect upon the potentials. Cyclohexylamine is a relatively strong base (pKb = 3.36). Users of these cation-selective electrodes should be aware of this marked interference by the CHA cation. ACKNOWLEDGMENT
Dr. T. Nihei of the Department of Medicine very kindly loaned us his Model 92-32 electrode for these experiments. RECEIVED for review, May 5, 1970. Accepted July 6, 1970. Financial support was provided by the Medical Research Council of Canada.
Trichloramine-Nitrite Reaction for Determination of Blood Ammonia Francisco R. Seta and Beatriz E. Tamagno Departamento de Bioquhica Clinica, Facultad de Ciencias Medicas, Universidad de Rosario, Santa Fe 3100, Rosario, Argentina
THEVERY LOW concentration of ammonia usually present in blood necessitates sensitive analytical methods. Because present methods require delicate equipment, extra handling, or varying volumns of sample, they are not always applicable in clinical laboratories. The procedure herein described combines Conway’s technique ( I ) with the particularly sensitive Zitomer-Lambert reaction ( 2 ) and it is well suited to routine analysis. EXPERIMENTAL
Apparatus. Lucite diffusion dishes were Conway Type 11. The rotating apparatus was a disk 32 cm in diameter, with holes drilled 46 mm deep, into which the dishes were lodged, rotating at 36 rpm in an 18” inclined plane. The thermoregulated bath was a Haake circulating bath regulated at 26 “C. A Beckman D U spectrophotometer was employed. Reagents. All reagents were prepared with ammonia-free water. Except for the hypochlorite, all chemicals were of reagent grade. (1) E. J. Conway, “Microdiffusion Analysis and Volumetric Error,” The Macmillan Company, New York, 1958. (2) F. Zitomer and J. L. Lambert, ANAL.CHEM., 34, 1738 (1962).
STANDARD AMMONIA SOLUTION.A stock solution containing 100 ppm of ammonia-N was prepared by dissolving 0.4719 gram of dry ammonium sulfate in 1 liter of ammoniafree water. This solution was diluted to bring the concentration to 1 pg per ml. SULFURIC ACID SOLUTION, H & 0 4 (0.01N) was kept in 2-ml ampoules. The contents of each ampoule was diluted to 3 ml with ammonia-free water. BUFFER SOLUTION, pH 5.20 + 0.01, was prepared from trihydrated sodium acetate (136 g) and 21.2 ml of glacial acetic acid per liter. This solution should be adjusted to the indicated pH and kept in 2-ml ampoules. The contents of each ampoule was diluted to 45 ml with ammonia-free water. These reagents were stored in ampoules to avoid the possibility of airborne contamination and diluted just prior to use. POTASSIUM CARBONATE SOLUTION 60 (WjV). Methanol (1 ml) was added to 9 ml of this solution before use. HYPOCHLORITE SOLUTION.Four-tenths ml of “Ayudin” per 100 ml of solution. “Ayudin” is commercial sodium hypochlorite which contains approximately 0.8 g of active chloride per 100 ml. SODIUM NITRITE SOLUTION.One-half gram in 100 ml of water.
ANALYTICAL CHEMISTRY, VOL. 42, NO. 12, OCTOBER 1970
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