Ammonia Determination and Sample Preparation for Mass Spectrometer by a Micro Diffusion Method S. W. MAYER, F. H. KELLY, and M.
E. MORTON
Radioisotope Unit, Veterans Administration Hospital, Long Beach, and School of Medicine, University of California, Lor Angeles, Calif.
and added to Kessler's reagent so that the reagent is finally diluted to 1 to 10. Then the increase in absorbance produced by the ammonia is measured spectronhotometrically. Ordinarily, a dozen diffusion bottles are rotated simultaneously for 30 minutes. The sulfuric acid (containing the diffused nitrogen-14 and nitrogen-15 as NH4+) from each of those bottles holding samples for mass spectrometer analysis, is transferred to the two-legged flask (6) used for reaction of ammonia with hypobromite to furnish the nitrogen gas a t the manifold of the mass spectrometer.
In order to analyze rapidly large numbers of samples of nitrogenous metabolic products and chromatographic fractions, a simplified, rapid diffusion method has been developed and extended to a range from 0.5 y to 10 mg. of nitrogen. The standard deviation in ammonia recovery determinations was 2% for quantities of 10 y or larger, and 0.2 y for quantities less than 10 y. The method is being used to prepare samples for mass spectrometer determinations, supplanting the Kjeldahl distillation method.
RESULTS
The results of a study of the effect of time of rotation on the recovery of ammonia are summarized in Figure 1. Complete recovery was attained in 20 minutes for 1-7 and 1-mg. samples of ammonia nitrogen. The solid circles in Figure 1 summarize the experimental data for the recovery of 1 mg. of ammonia nitrogen when the rotation was carried out a t 25' C., rather than a t the regular temperature of 65" C. At room temperature, a t least 40 minutes were required to obtain complete recovery. A series of measurements of the recovery of ammonia nitrogen in the range from 1 to 15 7 provided a linear calibration curve.
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N T H E course of an investigation of nitrogen metabolism with
nitrogen-15, it became necessary to develop a method for the determination of inicrogram quantities of nitrogen in several organic and inorganic materials. Inasmuch as such determinations of nitrogen frequently are resolved into determinations of ammonia, Conway ( 1 ) and Kirk ( 3 ) have devised ammonia diffusion cell methods involving titrimetry. Linderstrom-Lang and Holter ( 4 ) and Seligson and Seligson ( 5 )employed single cell microdiffusion arrangements in which the ammonia was absorbed in an acid solution suspended above the alkaline sample. The authors have modified the latter method to extend its range from 0.5 y to 10 mg. of nitrogen, and have employed the procedure as a timesaving means for preparing ammonia samples prior to isotopic analysis in the mass spectrometer. The method is rapid, utilizes readily available materials, and is so simple manipulatively that it is well adapted to the routine handling of large numbers of samples by technicians. PROCEDURE
Diffusion of the ammonia is carried out in a 30-ml. bottle (30 mm. in diameter, 70 mm. high). A one-hole rubber stopper (KO. 0) which holds a glass rod, seals the bot,tle. The base of the glass rod is 20 mm. from the bottom of t,he vessel and is slightly broadened so that t,he rod holds ten borosilicate glass "helices" which had been placed on the rod before the base of the rod had been fused and broadened. The helices serve to hold a relatively large quantity of sulfuric acid solution on the rod. For samples liberating milligram quantities of ammonia the rod is loaded with SOY0 sulfuric acid; 5y0 sulfuric acid is adequate for the microgram range. The sample is placed in the bottle; eight glass beads are added to facilitate mixing and diffusion, and enough distilled water is pipetted in to bring the liquid volume to 3.00 ml. Then 1.5 ml. of saturated potassium carbonate are added, and the bottle is immediately sealed with the stopper holding the glass rod. In order to expedite diffusion, the bottle is rotated for 30 minutes (with its asis horizontal) a t 45 r.p.m., while held in a clamp so t,hat it is 1.7 em. from the center of rotation. During the rotation two stationary infrared lamps heat the bottle to a temperature of 65" =t 2" C. The Bock-Benedict modification ( 2 ) of Kessler's reagent is used for t,he ammonia determination, n-ith the Beckman DU spectrophotometer at the 418 mp peak. For microgram quantities of ammonia, 3.00 ml. of the 1 t,o 10 (volume per volume of water) Sessler's solution are transferred to a 1-em. Cores absorption cell. The stopper is then removed from the 30-ml. diffusion bottle, and the base of the glass rod is put directly into the Sessler's solution in the Cores cell. The rod is moved vigorously up and down several times to secure efficient, rinsing and mixing action, 5 minutes are allon-ed for color development, and the absorbance of the solution is compared to that formed n-ith a suitable blank. For milligram quantities of ammonia, the 50% sulfuric acid solution which has absorbed the ammonia is washed from the rod into a volumetric flask. Suitable aliquots are then diluted
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ROTATION TIME, MINUTES
Figure 1.
Effect of time of rotation on recovery of ammonia
Several sets of twelve replicate determinations a t a single concentration were made in order to evaluate the reproducibility of the standard procedure. The mean recoveries of ammonia nitrogen and the corresponding standard deviations for 1.00 y, 10.0 y, and 1.00 ing. were, respectively, 1.04 i 0.18 y, 9.91 rt 0.217 and 0.999 =t 0.19 mg. Recoveries of 10.0 y of ammonia nitrogen added to blood or urine of known ammonia content were 10.1 + 0.22 y and 10.0 f 0.22 y, renpectively. DISCUSSION
Kirk ( 3 )has pointed out that the alkaline solutions in ammonia diffusion methods should have shallow liquid layers. Bccordingly, in the present procedure the horizontal position of the bottle, the use of glass beads, and the rotation of the bottle were designed t o facilitate diffusion. The use of heat lamps proved to be convenient for raising the temperature and increasing the rate of
ANALYTICAL CHEMISTRY
838 processes involved in thia method. When such heating would lead to significant hydrolysis of amides present, t h e diffusion should be carried out a t lower temperatures for correspondingly longer time intervals. The accuracy of the Sessler spectrophotometric method appears to be somewhat inferior to t h e within 1% accuracy observed in Conway’s and Kirk’s titrimetric procedures. The ease of manipulation in t h e present procedure, in which the color is developed directly in t h e absorption cell, is advantageous, however, when large numbers of microanalyses are to be run in a relatively routine way: The analyses of t h e large numbers of nitrogenoufi materials separated, for example, from chromatographic columns or from the products of tissue slice or homogenate metabolism could be facilitated by this simple procedure. The ammonia can be stoichiometrically produced (3) from urea, amides, amines, total nitrogen, and nonprotein nitrogen. The microdiffusion technique has proved to be highly satisfac-
tory and convenient for preparing nitrogen samples for isotope ratio determination with t h e mass spectrometer. The ammonia sample is obtained directly in an interference-free form for t h e usual treatment with hypobromite (6) t o produce nitrogen gas. LITERATURE CITED
Conway, E. J., and Byrne, A., Biochem. J . , 27, 419 (1933). Hawk. P. B.. Oser. B. L.. and Summerson. mi. H.. “Practical Physiological Chemistry,” 12th ed., p. 1230, Blakiston, Philadelphia, 1947. Kirk, P. L., “Quantitative Ultramicrcanalysis,” Chap. 7 , Wiley, Kew York, 1950. Linderstrom-Lang, L., and Holter, H., 2. physiol. Chem., 220, 5 (1933).
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Seligson, D., and Seligson, H., J . Lab. Clin. M e d . , 38, 324 (1951). Sprinson, D. B., and Rittenberg, D., ,Vaval Medical Bull., Supplement on Preparation and LIeasurement of Isotopes, p. 82, 1948. RECEIYED for review July 15, 1954.
Accepted November 12, 1Q.54.
Procedure for Determination of Diffusion Coefficients of Gases and Nongaseous Solutes for Membranes SVEND G. JOHNSEN and JOHN ESBEN KIRK Division o f Gerontology, Washington University School o f Medicine, St. Louis, M o .
A procedure was developed for determination of the diffusion rates of gaseous and nongaseous solutes from one fluid phase to another through biological or inanimate membranes. The procedure permits determination under a constant total pressure of the diffusion coefficients for several solutes within a single experimen t
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ROCEDURES for determination of diffusion rates of gases through membranes have been described by Krogh (5) and
Wright ( 5 ) . The apparatus devised by Krogh permits measurement of diffusion rates of oxygen and carbon dioxide in separate experiments, whereas the apparatus used by Wright is applicable only to the study of the diffusion of carbon dioxide. The procedure desiribed in the present publication represents a further development of Krogh’s diffusion method. It permits measurement of the diffusion rates of gases and nongaseous solutes from one liquid phase to another through biological or inanimate membranes, utilizing the principle for sample transfer and gas analysis employed by Kirk and Hansen (2). PROCEDURE
Apparatus. DIFFUSIONAPPARATUS.The details of the assembled diffusion apparatus are shown in Figure 1. The apparatus consists of two 50-ml. glass syringes (A. S. Aloe precision syringes): the bottoms of the barrels have been removed and the barrel tops have been ground plane. The membrane is interposed between the ground barrel tops of the syringes and is held in place by two metal diaphragms with circular openings and two hard rubber 1.25-inch slip joint washers. A cannula of stainless steel about 1 mm. in outside diameter (hypodermic needle, gage 19) is inserted through each rubber ring. The tips of the needles are cut off a t a right angle and do not extend beyond the inner surface of the rings. Airtightness of the perforation canal is ensured by sealing the place of entrance of the needle with cement (Sealit, Fisher Scientific Co.). The outer ends of the needles are closed by insertion of a oneway metal stopcock with two male outlets. Rotation of the diffusion apparatus is provided by an electric motor, as shown in the insert of Figure 1. A screw-shaped glass piece about 1 cm. in length, placed in each compartment, effects stirring of the solution during the rotation of the apparatus. The metal diaphragms used in the diffusion apparatus are
made of chromium-plated steel. Sets Kith beveled rircular openings 25, 20, 15, and 10 mm. in diameter have been found suitable. The rubber washers should be tested for gas permeability. With the hard-rubber washers used by the authors no measurable loss of gas was observed over a 2-hour period in experiments in which the compartments were filled with carbon dioxideaerated water and a steel plate interposed between the rubber rings. EQUIPMEXT FOR GASANALYSIS.The equipment for gas analysis includes one, or preferably two, Van Slyke manometric apparatus with extraction chambers provided with calibration marks a t 0.5-, 2.0-, 10.0-, and 50.0-ml. volumes. A stainless steel cannula 7 5 mm. in length and about 2 mm. in outside diameter (hypodermic needle, gage 15) with attached rubber tip is used for transfer of samples from the diffusion apparatus to the extraction chamber of the Van Slyke apparatus (2). Reagents. The reagents for gasometric determination of carbon dioxide, oxygen, and nitrogen are described by Kirk and Hansen (2). Technique for Use of Diffusion Apparatus. I n diffusion studies on animal membranes the experiments are preferably carried out under sterile conditions. After the membrane, the metal diaphragms, and the rubber washers have been inserted between the syringe barrels, the apparatus is screwed firmly together. The metal stopcocks and the outside parts of the needles are secured in a fixed position by means of thin copper wire. For evperiments on gas diffusion about 50 ml. of buffer medium are heated in a beaker t o 43” C., and the solution is aerated with the appropriate gas as described by Kirk and Hansen ( 2 ) . Thirty to 40 ml. of the solution are then poured into one of the compartments of the apparatus, the screw-shaped glass piece is placed in position, and the plunger is inserted. Anv free gas present is ejected, and the volume of solution introduced is determined by weighing. Buffer medium for the other compartment is then prepared and similarly introduced. The plungers require no special fastening, but n-ill stay in place during the rotation of the apparatus. The diffusion apparatus is finally placed horizontally in the belts of the rotator (see insert, Figure 1) in a thermostat a t 37” C., and rotation is started. It is convenient to place a shield of paper board or a rubber ring around each syringe barrel to keep the belts in place during the rotation. An elapsed time of 20 to 30 minutes is allowed to establish temperature equilibrium and initial penetration of the gas through the memhrane. The apparatus permits determination of the diffusion coefficients of several gases in the same experiment. Buffer medium aerated with oxygen may be used in one of the compartments,
