desirable to duplicate thi: determination a t different periods of decomposition time to ensure complete decomposition. Advantages of Method. -4 small amount of sample containing 0.5 to 1 mg. of boron suffices for analysisless than in the titrimetric method. The ambiguities in the determination, such as the choice of pH range in the titrimetric method, have been eliminated by converting boron into boric acid, which is identical to the standard substance, so that no calibration is needed against any interfering effect. I n the gravimetric d1:termination of carbon and nitrogen in organoboron compounds low values occasionally were obtained (4, 6), probably because of the formation of boron carbide, boron nitride, or both, in the course of combustion. If boron is determined simultaneously, the value m u d also be low, because of incomplete rligestion. The
present method of analysis does not have such handicaps. While slight modifications are needed in the procedure for preparing sample solutions for a few compounds, the general principle that boron is converted to boric acid is not altered, and naturally the method is applicable to compounds which do not contain nitrogen. The analysis is somewhat time-consuming, because of the comparatively long period for decomposing treatment, but that time can be utilized for other work. The time of manual operation is less than 1 hour for one run. To exhibit the accuracy and precision of the method, recoveries (per cent of required) are listed in Table 11. The mean error is 0.6 (per cent of required) and the relative standard deviation is 0.6i% with 22 results in Table 11. Thus the method gives satisfactory values for ordinary demands. If a larger amount of sample is available, a higher
precision can be obtained by using two or more additions of the standard boron solution. ACKNOWLEDGMENT
The author expresses appreciation to Haruyuki Watanabe, Toshio Nakagawa, and Ken’ichi Takeda for their helpful advice and encouragement. LITERATURE CITED (1) Buell, B. E., ANAL.CHEM.30, 1514 11968). ( 2 ) Ibzd., 34, 635 (1962). (3) Dean, J. .4., Thompson, C., Ibid., 27, 42 (1955). ( 4 ) Gerrard, W., Hudson, H. R., Mooney, E. F., J . Chem. SOC.1962,113.
( 5 ) Gilbert, P. T., Jr., Hawes, R. C., Beckman, A. O., ANAL.CHEM.2 2 , 772 (1950). (6) Watanabe, H., Totitni, T., Nagasai+:i,
K., Yoshizaki, T., Shionogi Research Laboratory, Shionogi & Co., L t d , Osaka, Japan, unpublished data, 1962. RECEIVEDfor review April 9, 1963.
Accepted September 9, 1963.
Spectrophotometric Determination of Nitrogen in Total Micro-Kjeldahl Digests Application of Phenol-Hypochlorite Reaction to Microgram Amounts of Ammonia in Total Digest of Biological Material LEWIS T. MANN, JR. laboratory o f Chemical Pathology, Department o f Pathology, Harvard Medical School, Boston 7 5, Mass.
b Modifications of putttished methods have permitted the direct estimation of ammonia in total micro-Kjeldahl digests by means of formation of “phenol-indophenol” from ammonia and phenol in the presence of base, sodium hypochlorite, and sodium ferrinitrosopentacyanide (“nitroprusside”). In the reported procediire, 1 to 15 pg. of nitrogen (as ammonia) can b e determined. The absorptivity of the colored solutions ranges from 1.1 to 1.4 X l o 3 cm.-l./gram ( E = ca. 15-20 X lo3)a t 630 mp, Absorptivity is constant with NH, concentration from 0.1 pg./mI. to 1.5 pg./mI. Once formed, colored solutions in this range obey the Beela-Lambert law, and those solutions, too dark for direct spectrophotometric obiiervation, may b e diluted to 2 to 5 volumes with an appropriate buffer without loss of linearity.
A
there are reports in the literature of the titrimetric or colorimetric determination of nitrogen as ammonia in total micro-Kjeldahl LTHOUGH
digests (3, 8, 9), each method has presented some particular difficulty when we have attempted to apply it to biological materials. The problems are principally those of available sample size and a wide variability of nitrogen content. In addition, we required a rapid method (24 hours or less) which did not involve the use of ultramicro equipment and techniques. Several recent reports (2, 4-6, 11, 14, i7,19)have been published in which the formation of “phenol-indophenol” from phenol and ammonia under basic oxidizing conditions has been used to determine ammoniacal nitrogen. The test is extraordinarily sensitive, so that under optimal conditions, amounts of nitrogen (as ammonia) in the microgram range should be determinable. Lubochinsky and Zalta (11) described uniquely a system in which the total Kjeldahl digests could be used in this colorimetric procedure. Because we experienced difficulty in repeating this procedure on our Kjeldahl digests, we undertook a more detailed investigation of the color reaction, under the conditions with which we had to contend,
namely, the use of a mercuric ion catalyzed Kjeldahl digest, which was adapted from Ogg (IS) and Steyermark (18). EXPERIMENTAL
Apparatus. A square aluminum block, 8 inches on a side, about 2.5 inches thick, was drilled with 36 evenly spaced holes 3//* inch in diameter, and 2 inches deep. To the underside of the block was fastened a 1000watt heating element (Calrod) which mas controlled by a variable transformer. Heat loss was cut by surrounding the block with glass wool. =i flat pan, filled with washed sand, placed atop a hot plate can also be used, but with less convenience. Reagents. All water was either distilled or passed over a sulfonic acid ion exchange column. Zinc dust, low X, finely divided, impalpable powder. Coarser grades (60- to 200-mesh) were not satisfactory. Sodium Phenolate Reagent. Phenol was distilled from aluminum turnings ( 7 ) and stored (tightly stoppered) in a refrigerator. Phenol (6.0 grams, 0.059 mole), was placed in a 100-ml. voluVOL. 35, NO. 13, DECEMBER 1963
2179
metric flask. To it was added about 50 ml. of ice cold water, followed by trisodium phosphate (3.8 grams, 0.01 mole) and sodium hydroxide (2.9 grams, 0.073 mole). The mixture was made up t o 100 ml. with water. Procedure. All standards, samples, and blanks were prepared in triplicate where possible. T o ensure parallel treatment of all samples, a single batch of water must be used throughout for washing and dilutions. All glassware must be base rinsed before use. Samples of homogenates and tissue fractions derived from biological material and, if possible, estimated t o contain from 1.0 to 10 pg. of nitrogen (0.07 to 0.7 micromole) were pipetted or weighed into heavy walled '(ignition" tubes (18 X 150 mm.). Digestion solution (18) (0.50 ml.) wac: pipetted into each tube, and an alundum or carborundum boiling chip (acid and base washed) was added to each tube. Appropriate reagent blanks and standards were prepared. The tubes were heated to 120" C. in a sand bath or block (as described) until most of the water was driven off, The temperature was then brought as rapidly as possible to 345" to 350" C., and maintained for 1 hour, after which the tubes: were removed to a rack to cool. If necessary, additional concentrated sulfuric acid (0.05-ml. volumes) was added with additional heating to complete the digestion. To each tube was added 1 ml. of n-ater, which was brought to a brief boil over a low flame to wash down the inner walls of the tube. When each tube had again cooled, zinc dust (about 25 to 50 mg.) was added on the tip of a tapered spatula. Immediately after the addition of zinc, each tube mas vigorously mixed ("snapped") to break up the "coagulated" mass of zinc dust. Each tube was again heated over a low flame and the contents were boiled vigorously for 20 to 40 seconds. The tubes were allowed to stand a t room temperature overnight. The tubes were then cooled and maintained in a cold water (3" to 5" C.), 0.01% aqueous phenolphthalein (0.1 ml.) was added to each tube, the contents of which were subsequently titrated to a permanent pink with approximately 0.4N sodium hydroxide (3.5 to 4.5 ml.). The volume of titrant for each tube was noted, and sufficient water was added to bring the contents of each tube to 6.0 ml. Into each tube (still in the cold water
Table I.
RESULTS A N D DISCUSSION
Digestion Procedure. Although we did not study the digestion procedure extensively, we did observe low results when either copper or selenium dioxide was used as the catalyst. The relative efficiencies of selenium and mercury have been investigated extensively by Baker (I), Steyermark (18), and Ogg ( I S ) , and they have observed the loss of ammonia when selenium wa3 used. For convenience, we prepared a digestion solution containing sulfuric acid, potassium sulfate, and mercuric sulfate (added as mercuric oxide) and have added in a single aliquot the reagents added separately by Steyermark (18). As would be expected, failure to remove the mercuric ion after digestion reduced the absorptivity after color development of the final mixture almost t o zero. Seither sodium thiosulfate nor sodium borohydride vas as effective in removal of the catalyst as zinc dust, judged by a decreased absorptivity of the final mixture. (Sodium thiosulfate was used as directed by Steyermark (18); sodium borohydride was added to the cool diluted digest as 0.1 ml. of 0,4'% aqueous solution.) Because zinc ion is precipitated by base, we investigated its possible interference in the final step of the analytical procedure. Surprisingly, the only difficulty we observed was a result of the
Absorptivity of Final Solution a s Functions of Ferrinitrosopentacyanide Concentration and Time Development temp. = 40" C.
Ferrinitrosopentacyanide
concentration
4 . 2 X 10-4M(1+8000) 5 . 6 X l O - ' M (1 -+ 6000) 8.4 X 1 0 - 4 M ( l +4000)
21 80
bath) was pipetted sodium phenolate reagent (1.0 ml.), followed by thorough mixing of the contents of each tube. The pipetting of an aqueous solution of sodium ferrinitrosopentacyanide (4.2 x 10-4M, 1.0 ml.) was also followed by thorough mixing. The final reagent, aqueous sodium hypochlorite solution (0.015M, 2.0 ml.), was added in a like manner. The tubes, closed with stoppers, were placed in a covered 40" C. water bath for 30 minutes and were subsequently cooled for about 5 minutes in a cold tap mater bath. The precipitates of zinc hydroxide were sedimented by brief centrifugation. The absorbances of the solutions were determined at a wavelength of 630 mp against a distilled water blank, and the amounts of nitrogen in the unknowns are determined from a calibration curve in the usual may.
Absorptivity 10 0 092 0.102 0 110
ANALYTICAL CHEMISTRY
at stated
20 0.139 0.137 0.135
time (expressed/pg. /ml.) minutes
30 0.146 0.139 0.135
60
90
135
0.143 0.138 0.135
0.142 0 138 0.134
0.141 0.136 0 133
effectiveness of zinc ion as a buffer between p H 8 and 9. The indeterminate amount of zinc which enters solution as a result of reaction with the digestion solution required use of a n indicator which will permit titration beyond the buffering range of zinc ion but which will not absorb light significantly at 630 mp. Phenolphthalein and Nile blue A are both effective. The latter, changing from blue to pink, has a somewhat higher pK and, therefore, will allow neutralization to proceed somewhat further than phenolphthalein. Both ha\-e been used successfully. K e have not been fully successful so far in decreaqing the time required for the removal of mercury. Since mercury metal is attacked by hot 2N sulfuric acid (the approximate concentration of the diluted digests), we have added sufficient sodium acetate (1M) to neutralize the sulfuric acid before adding zinc. Although the absorptivities of the solution are fairly conitant, they are significantly lower than those of solutions which have been allon ed to react overnight with zinc. Color Development. T o study the phenol-hypochlorite-ammonia color step by itself, we omitted the digestion step and used 0, 2, 5 , and 10 pg. of nitrogen (as ammonium sulfate) to cover a reasonable range of nitrogen concentration. To the standard ammonium sulfate in 7 ml. of aqueous solution was added an appropriate dilution of the reagent under study in 1.0 ml. Except where the other order of reagent addition was purposely changed. the reagents were always added in the order of phenol, ferrinitrosopentacyanide, and hypochlorite. Except when time mas the variable, the colors which developed in 60 minutes were estimated a t 630 mp. According to Llusso and Beecken (IW),phenolindophenol in methanolic potassium hydroxide shows four absorption peaks, the largest of which lies a t 635 to 640 mp ( E = 39.8 X lo3). K e have found a very 4milar maximum (630 to 635 mp) in aqueous base. (We could not verify the other maxima with our preparation because of interferences by ferrinitroaopentacyanide ion and the large excess of phenol.) K h e n the phenol concentration was varied between 0.54X and 1.8M, the absorptivity remained constant, but it decrea-ed a t concentrations below 0.54M. The latter figure is equivalent to 5 grams of phenol in 100 ml. of reagent. as described by Lubochinsky and Zalta (21); iince it is close to the lower limit giving maximal absorptivity, we have increased the concentration to 6 grams per 100 ml. An increase of concentration to 2.7M (25 grams/100 ml.) [Russell (I:)] decreased the absorptivity. Hypochlorite was derived from reagent sodium hypochlorite or Cloros, determined to be 0.754 and 0.777M, respectively, by iodometric titration. Maximal absorptivity was observed in a concentration range in the reaction
miiture of 0.002 to 0 004X. A comparison of Clorox with reagent sodium hypochlorite showed the tFTo to be completely interchangezble. Varying the concentration of sodium ferrinitropentacyanide in the reaction mixture produced three effects. At the higher concentration (16.8 X 10-5M) the absorbance (and t ierefore presumably the amount of "phmolindophenol") FT as smaller than a t ower concentration (4.2 X 10-5JI) However, the final color n a s stable for longer time periods a t high concentrations than a t low.,and the final absorptivity was more rapidly attained at the higher concentrations (Table I). The effect of varymg pH (sodium hydroxide concentration) was observed by preparing a phenolate reagent omitting sodium hydrclxide (but adding trisodium phosphate) One milliliter nas added to each of the standard amounts of nitrogen (as ammonium ion), contained in 1.0 ml. Varying amounts of 0.2N scdium hydroxide were added t o the series of tubes; prior to addition of t i e final two reagents, the volumes of the mixtures were adlusted to 8.0 ml. v;ith water. The results are shonn in Table 11. There was a definite maximLm a t pH 11.4 to 11.6. The absorplivity decreased slowly as the pH was increaqed beyond 11.9. The pH was measured n i t h a t l p e E-2 glass electro le, for which the sodium ion correctio 1 was negligible (
