For a mixture of reactant and sample in the sources of the mass spectrometer, two competitive electron impact ionization processes can occur : Reactant & Ions (He+, or CHI+, etc.)
(2)
Sample $- Ions
(3)
The relative importance of each of the reactions is determined by the electron impact cross section for ionization. The cm2; ionization cross section for He is about 0.4 X cm2(11, 12). CH4, 4.7 X 10-le cm2; and acetone, 10 X Since the ratio of ionization cross sections of sample/He is much greater than the ratio of cross sections of sample/CH*, direct ionization of the samples will be more likely with He as a carrier gas than with CHI. The spectra of the same compounds obtained with the two reactant-carrier gases, He and CHI, are very different from each other as may be seen by comparing the spectra for methanol in Tables I and 11. CHI as a carrier gas gives ions resulting from proton and hydride transfer and subsequent decomposition, (MW l)+, (MW - I>+, and lower frag-
+
(11) F. W. LamDe, J. L. Franklin, and F. H. Field. J. Amer. Chem. soc., 79,612911957). (12) J . A. Beran and L. Kevan, J. Phys. Chem., 73,3866 (1969).
ment ions. He as a carrier gas gives ions resulting from predominantly electron impact ionization or charge transfer, MW+, and fragment ions. The work done with methane on well resolved and composite chromatographic peaks was repeated with helium as the carrier gas and comparable results were obtained. Further work on these systems which is planned includes studying the reproducibility of the system, the effects of operating parameters, and the possibility of quantitation. ACKNOWLEDGMENT
Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society and to the University of Delaware Research Foundation for partial support of this research. One of us (DMS) is grateful to the Sun Oil Co. for a leave of absence and support during part of this research. The use of this method is covered by patents pending by Frank Field and M. S. €3. Munson. This patent is owned by Esso Research and Engineering Company with. exclusive manufacturing rights to Scientific Research Instruments Corp,, Baltimore, Md.
RECEIVED for review June 23, 1970. Accepted September 14, 1970.
Determination of lood Ammonia by
pectrometry
Robert N. Hager, Jr.,1 David R. Clarkson,* and John Savorys Department of Aerospace Engineering, Unicersity of Florida, Gainesville, Fla.
A VARIETY OF METHODS have been described for the measurement of ammonia in blood. Most of the procedures have involved a preliminary separation of ammonia from blood either by diffusion (1-8) or by ion exchange chromatography (9,IO). Following this separation the ammonia has been determined by volumetric titration ( I , 6, 8)) coulometry (5), or colorimetry (3, 4 , 7-10). Okuda et al. (11) measured ammonia directly on a protein free filtration of blood using an indophenol colorimetric method, and some modifications of this procedure have been reported (12, 13). Enzymatic 1 Present address, A.B.A. Industries, Inc., P.O. Box 517, Pinellas Park, St. Petersburg, Fla 33565 a Present address, University of Pittsburgh Medical Center, Pittsburgh, Pa. a Present address, Pathology Department, University of Florida, College of Medicine, Gainesville, Fla.
(1) E. J. Conway and R. Cooke, Biockem. J . , 33, 457 (1939). (2) E. J. Conway, “IMicrodiffusion Analysis and Volumetric Error,”
5th ed., Crosby Lockwood, London. (3) D. Seligson and K. Hirahara, J. Lab. Clin. Med., 49, 952 (1957). (4) P. V. D. Burg and H. W. Mook, Clin. Chim. Acta, 8,162 (1963). ( 5 ) G. D. Christian and F. J. Feldman, ibid., 17, 87 (1967). (6) B. A. Tobe, Can. Med. Ass. J., 84, 767 (1961). (7) A. F. K. Buys Ballot and C. Steendijk, Clin. Chim. Acta, 12, 55 (1965). (8) S. Gangolli and T. F. Nicholson, ibid., 14, 585 (1966). (9) D. J. Foreman, Clin. Chem., 10, 497 (1964). (10) S. G. Dienst and B. Morris, J. Lab. Clbt. Med., 64, 495 (1964). (11) H. Okuda and S. Fujii, Saishin Igaku, 21, 622 (1967). (12) H. McCullough, Clin. Chirn. Acta, 17,297 (1967). (13) H. Leffler, Amer. J. Clin. Parhol., 48, 233 (1967).
procedures applied also to protein free filtrates have been described (14,15). The method described here involves the technique of derivative spectrometry and differs from all previous methods in that a measurement is made directly on molecular ammonia. Ammonium ion is converted to ammonia by adjusting the sample to pH 11. At 25 “C,total ammonia nitrogen then consists of 98% ammonia and 2 % ammonium ion. The ammonia dissolved in the sample contained in a closed vessel establishes an equilibrium with ammonia in the vapor state. At low concentrations, the equilibrium vapor pressure is proportional to the concentration of dissolved gas in the fluid in accord with Henry’s law. The ammonia vapor is measured using ultraviolet derivative spectrometry. The theory and operation of derivative spectrometers have been described previously (16-18). The second derivative spectrometer used in the present study has a single light beam which passes through the analyzing cell and is directed upon a radiation detector. The wavelength of this beam varies sinusoidally in time about a center value, the amplitude of wavelength modulation being approximately equal to the width of an ammonia absorption band (typically 20
A).
(14) K. L. Reichelt, E. Kvamme and B. Tveit, Scand. J. Clin. Lab. Inaest., 16, 433 (1964). (15) M. Rubin and L. Knott, Clin. Chim. Acta, 18,409 (1967). (16) E. C. Olson and C. D. Alway, ANAL,CHEM.,32, 370 (1960). (17) G. Bonfigliolig and P. Broveto, Appl. Opt., 3, 1417 (1968). (18) D. T. Williams and R. N. Hager, Jr., Appl. Opt., 9, 1597 (1970).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 14, DECEMBER 1970
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solution (0.05 ml) containing 100 pg per ml NH3 was added and the peak height (h3)recorded. The total ammonia content of the mixture prior to standard addition was computed by the formula:
lope of Regression
The ammonia content of the reagent blank was also determined using the same formula by adding 0.5 ml of water and 0.5 ml of trichloracetic acid solution to a dean cell containing 0.8 gram of potassium carbonate. The total ammonia contained in 1 rnl of whole blood was determined by: (AM)biood = 2{(AM)~ernatant- (AMhsnkI pg/ml
(2)
RESULTS AND DISCUSSION I.o 2.0 30 4.0 5.0 AMMONIA (pg/ml) DERIVATIVE SPECTROMETRIC PROCEDURE
Rapid changes occur in the ammonia concentration of untreated blood samples. In order to prevent such changes, Figure 1. Comparison data between a colorimetric a trichloracetic acid filtrate of whole blood was prepared a diffusion procedure and the derivative spectrometric few seconds following venipuncture and the low pH of this procedure filtrate prevented loss of ammonia during storage. Blood treated in this way with the filtrate stored at -4 "C was found to be stable for at least 96 hours. When the center wavelength is set to coincide with the center The addition of potassium carbonate to the protein free of an absorption band, the signal from the detector consists filtrate increased the pH to 11, thus converting virtually all of a steady mean value with an ac signal superimposed, the amonium ion to ammonia, and also produced a "salting out" amplitude of which is proportional to the difference between effect which decreased the solubility of ammonia in the solulight absorbed by the ammonia vapor and unabsorbed light. tion. The calibration curve of derivative spectrometer peak This ac signal is separateIy amplified, using a tuned amplifier height us. ammonia concentration was linear to 10 pg of which rejects all other signals due to receiver noise, etc., and ammonia per ml. The precision of the blood ammonia the amplified signal is then divided electrically by the mean procedure was determined by performing duplicate determinadc signal suck that the quotient is independent of any variation tions on 33 blood samples. The mean concentration was of the light source intensity. 2.26 pg/ml with a range of 8.66-5.22 and a standard deviation E X ~ ~ ~ I ~ ~ E ~ ~ A of~ 0.22 pg/ml. The mean recoveries of 2.0 and 10.0 p g of ammonium ion added to 1 ml of blood having a concentration Reagents. High purity trichloracetic acid in aqueous of 8.86 pg/ml were 90% and 108%, respectively. Determisolution (10 % w/v) and reagent grade anhydrous potassium carbonate were employed. A standard solution containing nations of blood ammonia levels by this derivative spectro100 pg per ml of ammonia was prepared by dissolving 0.3897 metric method were compared with measurements by the gram of ammonium sulfate in 1 liter of water. This solution diffusion procedure of Seligson and Hirahara (3) using the was further diluted to yield working standards. colorimetric reaction described by Chaney and Marbach Apparatus. The derivative spectrometer consists of a (19). The comparison data for 20 blood samples are given meter grating monochrometer modified to accept an osciilatin Figure 1. The slope of the regression line was 0.87 with a ing entrance slit and an analyzing section located between standard error of estimate of 0.53 pg/ml. The ammonia the stationary exit slit and the photomultiplier tube. The level found in 15 healthy laboratory workers was 1.13 total output current of the photomultiplier tube was amplified 0.69 pg/ml (95% confidence limits) with a range of 0.66by an ac-dc amplifier stage and the ac portion was further 1.75 pg/ml. amplified and rectified within a phase lock-in amplifier circuit. The rectified ac voltage was divided by the mean dc voltage The data presented in this paper indicate that the derivative and displayed on a potentiometer recorder. spectrometric method provides blood ammonia measureThe absorption cell, which was placed within the analyzing ments comparable to those obtained using spectrophotosection of the derivative spectrometer, contained quartz metric diffusion procedures. Furthermore, a physical propwindows and was 22-mm 0.d. and 19-mm i.d. with a 100-mm erty of molecular ammonia i s detected directly, Le., the ablight path. sorption of ultraviolet radiation over discrete bands of waveProcedure. Five milliliters of blood was drawn without length. Because the absorption spectrum for ammonia stasis and immediately added to 5 ml of 10% cold (4 "C) consists of a unique array of bands and since these band locatrichloracetic acid. The mixture was shaken vigorously, tions and relative intensities can be readily observed during centrifuged, and the clear supernatant decanted and stored a blood ammonia determination, this method of detection is at -4 "C. One such sample supplied specimens for single and duplicate measurements and for supernatant storability specific for ammonia, once the vapor has been released from tests. For a single measurement; the absorption cell was the biological sample. cleaned with 50% HNQp, rinsed with ammonia-free water, and dried. Anhydrous potassium carbonate 0.8 gram and 1 nil of the blood filtrate were added to the cell and the cell RECEIVED for review April 6, 1970. Accepted July 31, 1970. ports were closed. The solutions were mixed and all inside Work supported in this part by the Department of Health, surfaces of the cell were wetted. An instrument background Education, and Welfare, Grant APOO591. readhg (hl) was recorded before positioning the cell, and the 2 0 5 0 4 band height ( k z ) waS recorded over a period of 5 minutes with the cell in place. Standard ammonium sulfate (19) A. L. Chaney and E. P. Marbach, C h . Chem., 8, 130 (1962). 14
*
ANALYTICAL CHEMISTRY, VOL. 42, NO. 14,DECEMBER 1970
