are somewhat iveaker than the waterto-water hydrogen bonds. EXPERIMENTAL
The S l I R spectrometer was a Varian Associates Model T'4300-B with a sample spinner, a 12-inch electromagnet, and a Model YK3506 superstabilizer for the magnetic field. -411 spectra were obtained a t 22' to 24" C. with the spectrometer operating a t 40 Me. The magnetic field was varied through resonance with a Varian Model T'K3507 sloiv sweep unit. The spectra vvere recorded on a Varian GI0 recorder that was calibrated at the time of each measurement by a sideband technique ( 3 ) . The recorder calibration required frequencies from 80 to 120 c.P.s., which were provided by a Hewlett-Packard Model 200CD audio oscillator whose output was monitored by a Hewlett-Packard Model 522B frequency counter. Samples n-ere prepared by diluting water-saturated tributyl phosphate (c. P., Eastman) with dry tributyl phosphate or by adding water to dry tributyl phosphate. The samples n-ere standardized by Karl Fischer titration. The sample vials used in the spectrometer were made from 5-mm. borosilicate glasq tubing. The chemical shifts, Av, were defined as the separation in cycles per second of the water resonance from the methyl proton resonance of tributyl phosphate. Av is negative for applied fields less than that for the methyl resonance. On this scale the proton resonance for pure water occurs a t approximately - 160 C.P.Q. DISCUSSION AND RESULTS
Typical tributyl phosphate spectra are displayed in Figures 1 and 2 . I n
both figures the large, sharp peak a t the extreme right of the recording trace is due to the methyl protons. The broad, partially resolved hump to the left of this peak arises from most of the methylenic protons, nhile the quartet at the left of the trace comes from those methylenic protons that are attached to the carbon atoms immediately adjacent to oxygen atoms. With tributyl phosphate containing 1.7 weight water, the water peak occurs approximately midway between the t\vo methylenic proton groups (Figure 1). Figure 2, the spectrum for nearly saturated (6.2 weight 70 water) tributyl phosphate, shon s the water proton signal coincident n ith one line of the quartet. In this case the signal has both increased in intensity and shifted to a loner field strength. The chemical shift of the n ater proton resonance nith respect to the methyl peak from tributyl phosphate n a, ' measured for 11 samples whose n-ater concentrations ranged from 0.44 to 6.3 n eight 7c. At least five determinationwere made on each sample. The results. nhen plotted, show a linear relationship betn een the chemical shift and the concentration of nater, nith a slope of 4.9 c.p.s. per per cent nater. The precision of individual measurements was 0.65 c.p.s. ( n = 59, p = 95), M hich n as equivalent to 0.13 weight yowater. This method of determining the water content of tributyl phosphate is nondestructive and requires only 2 minutes per determination once the spectrometer is set up. As the technique does not involve intensity measurements, the stability and linearity of the radio-
frequency amplifiers have no effect on the results. The peak height or the area under the proton resonance peak also can be used as a measure of water concentration, but the results are less accurate by about a factor of five. The method is limited, in general, to a two-component system. The presence of nitric acid or any compound that either complexes the tributyl phosphate or reacts with the water would make the analysis invalid because such reactions also shift the position of the nater peak. LITERATURE CITED
S. S., Healyt T. V., Kennedy, J., McKay, H. A. C., Trans. Faraday SOC. 52, 39 (1956). ( 2 ) Ann. Rea. Phys. Chem., annual reviews of nuclear magnetic. resonance spectroscopy published since 1954. (3) Arnold, J. T., Packard, 11. E.. J . Chem. Phys. 19, 1608 (1951). (4) Callis, C. F., Van Kazer, J. R., Shoolery, J. N., ANAL. CHEV.28, 269 (1956). (5) Cohen, A. D., Reid, C., J . Chenz. Phys. 24,790 (1956). 16'1 Elsken. R. H.. Shaw. T. 31.,ASAL. CHEW27,290 (1955). ' (7) Geddes, A. L., J . Phys. Chenb. 58, 1062 (1954). (8) Gutowskv, H. S., Saika, A., J. Chem. Phys. 21,1688 (1953). (9) Huggins, C. &I., Pimentel, G. C., Shoolery, J . S . , J . Phys. Chem. 60, 1311 (1956). (10) Ogg. R. A., Helv. Phys. Acta 30, 89 (1957). (11) Reilly, C. A,, ASAL. CHEY.30, 839 (1958). (12) Shuler, W, E., E. I. du, Pont de Semours & Co., Savannah River Laboratory, .liken, S. C., private communications. RECEIVEDfor review August 13, 1958. Accepted October 23, 1958. Information developed during work under contract AT(O7:2).-1 with the U. S.Atomic Energy Commission. (1) Alcock, K., Grimley,
\
I
Microdetermination of Ammonium and Protein Nitrogen GORDON FELS and ROGER VEATCH Radioisotope Service, Veterans Administration Hospital, Hines, 111.
b A micromethod for the colorimetric determination of protein nitrogen is described for the estimation of 1 y as ammonium nitrogen with quantitative accuracy. The use of 0.5M citrate buffer stabilizes the color against pH effects and minimizes the neutral salt effect.
N
is used as a n analytical reagent for the colorimetric determination not only of amino acids ( 5 ) but also for other amine derivatives (3). Because the reaction takes place IXHYDRIK
equally n ell with ammonia, resulting in the same product (5), it suggests that ninhydrin could be used with advantage in this instance. Boissonnas and Haselbach (1) have utilized the reaction for the determination of protein nitrogen but demonstrated no quantitative recovery. The sensitivity of the colored reaction product t o p H and salt concentration would make such evidence mandatory, especially following protein digestion. Modifications of the original procedure (5) are presented which overcome these obstacles.
EXPERIMENTAL
Reagents.
Sinhydrin
reagent,
1 . O M citrate buffer, 26.26 grams of citric acid (CsHsOi.H20), and 0.4 grams of tin chloride dihydrate. (SnC17. 2H20) are dissoh-ed in 245 ml. of 1 S sodium hydroxide. Thc p H i, adjusted to 5.0 with several drops of 1 0 s sodium hydroxide and the total volunic. is brought to 250 ml. with distilled water. Four grams of reagent grade ninhydrin are dissolved in 100 nil. purified methyl Cellosolve. Standard aiiimonium sulfatc. Exactly 94.3 mg. of dried reagent grade ammonium sulfate are dissolved in 100 VOL. 31, NO. 3, MARCH 1959
451
,
ml. of 0.2N sulfuric acid. A 1 to 50 dilution of this stock solution provides 4 y of ammonium nitrogen per ml. for the standard curve. Preparation of Sample. One milliliter of the protein solution containing 50 to 150 y of nitrogen is added to a 30-ml. Kjeldahl flask which has been calibrated to 50 ml. on the flask neck. The digestion is carried out by boiling with 1 ml. of 50% sulfuric acid and 3 to 4 drops of 30% hydrogen peroxide for 3 hours. Five drops of the usual copper sulfate-potassium sulfate mixture [5 grams of copper sulfate pentahydrate (CuS04.5Hz0, 0.5 gram of potassium sulfate) in 100 ml. of water] may be added for resistant proteins, although the addition did not influence the results with the materials under investigation. After cooling, the volume is brought up to about 20 ml. with distilled water. Two drops of 0.1% methyl red indicator are added, followed by 1.5 ml. of 10N sodium hydroxide. Neutralization to the methyl red end point is completed with 1N sodium hydroxide. The final volume is brought up to the 50-ml. mark. Stock standards containing 50 to 150 y of nitrogen in the form of ammonium sulfate as well as a reagent blank,
Table I. Determination of Nitrogen in Representative Substances
Nitrogen, Substance Alanine
Theor. 15.7
Phenylalanine 8.48
ReFound covered 16.1 102 16.3 103 15.9 101 16.5 105 15.6 99 Av. 102 8.48 100 8.68 102 8.74 103 8.87 104 8.61 101 8.74 103 Av. 102
Bovine serum albumin 16.07 (3) 16.4 16.4 15.8 Lysozyme
18.6 (4)
102 102 98 Av. 101 18.0 97 18.7 100 17.9 96 Av. 98
Table II. Neutral Salt Effect in Presence of Different Citrate Buffer Concentrations
Absorbancea Buffer Concn., M
DqpresNo 5% sion, salt NatSOl yo 0.044 0.036 18.2 0.1 0.2 0.162 0.145 10.5 0.5 0.260 0.239 8.1 4 Reaction mixture contained 2 y of ammonium nitrogen.
452
ANALYTICAL CHEMISTRY
are carried through the same process as the protein sample. One-milliliter aliquots of the digested samples are transferred in triplicate to 125 X 15 mm. borosilicate glass test tubes. Two milliliters of freshly prepared ninhydrin reagent (equal volumes of 1.OM citrate buffer and 4% ninhydrin in methyl Cellosolve) are added and the tubes covered with aluminum foil. They are then placed in a vigorously boiling water bath for exactly 20 minutes and cooled immediately in 15’ to 20” C. water. Five milliliters of 1-propanol-water (1 to I) are added to each tube and the contents mixed by inversion and transferred to 105 X 18 mm. cuvettes. The absorbance is read in the spectrophotometer a t 570 mp.
0 5 M CITRATE BUFFER
MICROGRAMS OF AMMONIUM NITROGEN
Figure 1. Effect of buffer concentration on color development
RESULTS
The results with four representative substances-alanine, p h e n y 1a1a n i n e , crystalline bovine serum albumin (Armour & Co.), and crystalline lysozyme (Nutritional Biochemicals Corp.)-are given in Table I. Replicate determinations demonstrate the quantitative accuracy for the amino acids. The value of 16.2% nitrogen obtained for bovine serum albumin is in good agreement with the value of 16.07% already documented (6). Similarly, the value of 182% nitrogen for lysozyme compares favorably with the reported value of 18.6% (6). It is believed that the slight discrepancies can be attributed more to the differences in the preparations than to procedural error. DISCUSSION
The essential difference between the present procedure and that given by Moore and Stein (6) is the use of a concentrated citrate buffer. The marked increase in sensitivity from the use of 0.5M citrate buffer [rather than with 0.1M (6)j is demonstrated in Figure 1. The spectral absorbance curve of the colored reaction product obtained under the new conditions is not altered, so that absorbances may still be read a t 570 mp. The increased sensitivity in 0.5M citrate may be explained in part by the enhanced buffering capacity, and also by the ability of citrate to bind metal ions which interfere with the color development. Recently, Meyer (4) has used (ethylenedinitri1o)tetraacetate to eliminate the interference of metal ions. The depression of color intensity by neutral salts is likewise influenced by the concentration of the buffer. Table I1 gives the percentage depression of the absorbance in the presence of 5% sodium sulfate and different citrate buffer concentrations a t pH 5.0. The concentration of 5y0 mas chosen because
this is the approximate salt concentration after neutralization. The use of 0.5M citrate reduces the salt effect markedly, and by carrying the standards and reagent blank throughout the procedure, this error is quantitatively eliminated. It would appear from the above results that the salt effect influences both the indicator (colored reaction product) and buffer equilibrium, the latter directly through a depression of the hydrogen ion activity coefficient, and the former indirectly through the resulting pH change. Although the present procedure does not have the precision of the classic Kjeldahl determination of nitrogen, it is more sensitive and less laborious. I n this laboratory it has proved more satisfactory in accuracy and reproducibility than Kessler’s method for nitrogen. LITERATURE CITED
(1) Boissonnas, R. A., Haselbach, C. H., Helv. Chim. Acta 36, 576 (1953). (2) Fevold, H. L., Advances in Protein Chem. 6, 230 (1951). (3) Lea, C. H., Rhodes, D. N., Biochim. et Biophys. Acta 17, 416 (1955). (4) Meyer, H., Biochem. J . 67,333 (1957). (5) Moore, Stanford, Stein, W. H., J . Biol. Chem. 176, 367 (1948). (6) Tristam, G. R., (‘The Proteins,” Vol. IA,p. 215, Academic Press, New York,
1953.
RECEIVED for review July 2, 1958. Accepted October 2, 1958. Division. of Analytical Chemistr 134th Meetmg, ACS, Chicago, Ill., Spternber 1958.
Errata Sheets of errata for 1958, arranged so that they may be clipped out and pasted over the incorrect material, are available without charge from the Reprint Department, American Chemical Society, 1155 Sixteenth St., N.W., Washington 6, D.C.
