Precise Method for Determining Ammoniacal Nitrogen in Eggs ARTHURW. THOMAS AND MARGUERITE A. VAN HAUWAERT, Columbia University, New York, N. Y.
F
OR about 25 years i t has
,
water which would be displaced An apparatus which renders possible the prefrom one bottle to another in the been k n o w n t h a t hens' cise determination of ammoniacal nitrogen by same time. eggs will evolve traces of aeration of alkaline mixtures containing very ammonia upon aeration in the Using these methods, checks small amounts of ammonia is described. cold, after the addition of a small might be obtained in parallel I t is essential that the volume of air be measamount of alkali carbonate. The measurements using t h e s a m e ammonia so liberated has been air pressure and volume, but reured. Twelve hundred liters of air passed at a r e f e r r e d to as ammoniacal or sults could not be d u p l i c a t e d rate of 240 liters per hour are suficient to remove loosely bound nitrogen. The from day to day, The present quantitatively the loosely bound nitrogen from e x a c t m e a s u r e m e n t of t h e investigation was undertaken egg samples containing from 1.3 to 6.1 mg. of amount of loosely bound nitrowith the object of developing a loosely bound nitrogen per 100 grams of sample, gen recoverable from eggs is acprecise method for the quantita. cepted as perhaps the best means tive determination of l o o s e l y using the apparatus and procedure developed in for grading edible eggs, either as bound nitrogen in eggs and in this investigation. shell eggs direct from the nest or whole egg magma. Operating at I n order to remove quantitatively the loosely from cold storage, or in the form room temperature, the variables bound nitrogen from eggs, in a reasonabb and of frozen egg magmas. studied were rate of aeration, analytically feasible time, the initial p H of the Since distillation of alkalinized volume of air, concentration of egg magma cannot be used, owthe aerated alkaline mixture and alkalinized egg mixture should be at least 9.5. ing to the danger of decomposing influence of amount of alcohol At p H values higher than 10.7 a continuous dethe complex nitrogenous comtherein, and the p H of the alkacomposition of nonvolatile nitrogenous matter pounds present, an application line m i x t u r e . T h e n e e d for results, yielding ammoniacal nitrogen. of the Folin aeration method (4) such a study has recently been for the determination of amasserted by Alfend (1). monia in urine has been used for several years in the examinaAERATIONAPPARATUS tion of frozen eggs. It consists essentially in making the sample alkaline, removing the liberated ammonia by aeration, For the purpose of providing a flow of air under fairly conand absorbing it in standardized sulfuric acid solution. stant pressure, and measuring the volume of air involved in t h e The method as now used is not satisfactory because the aeration of each individual alkaline mixture, the apparatus operating conditions, chiefly with respeict to the volume of air shown in Figure 1 was developed. required and the rate of aeration, are not well defined. Until It was found necessary to introduce a pressure regulator into now the nearest approach to a quantitative prescription for the system. This regulator consisted of two concentric cylinders the conditions of aeration has been the following: of galvanized iron, each with one end closed. The outside cylinder, with the open end upwards, had a height of 47.5 inches (121 Hendrickson and Swan (6) advised the use of an air pump de- cm.) and a diameter of 8 inches (20 cm.), and was filled with livering a pressure of 14 cm. of water. A water gage is connected water to such a height that at the required speed of aeration, air with the aeration apparatus, keeping the air pressure constant was continuously escaping from the pressure regulator. The and thus insuring the passage of an equal volume of air through inside cylinder was 51.5 inches (131 om.) high and 2 inches (5 the aeration cylinder in a given time. Redfield (14, 15) used an cm.) in diameter. Its top was connected to the air line. It was air pump furnishing a blast with a pressure of 10 pounds per provided with rectangular holes, B, on the lower end to allow the square inch and discharging into a tank of sufficient size to com- excess air above the required pressure to escape through the water pensate for pulsations of the pump and deliver a steady flow. contained in the outside cylinder. This system gave very satisHe also insisted on running a blank experiment to determine the factory results. An increase of more than 3 to 4 mm. in the difpercentage recovery of ammonia, using a known amount of pure ference of water level in the flowmeters was seldom noticed over ammonium sulfate (containing about 3 mg. of nitrogen) and 25 a period of 6 hours of aeration at 240 liters per hour. Flowmeters placed between the air line and the aeration cylincc. of water. The recovery should be over 95 per cent to be deemed satisfactory. In subsequent reports by Marsh (11), ders made it possible to evaluate the volume of air passing through Lourie (9), and Hertwig (7) no details were given concerning the each individual cylinder in a given time. The inside diameter volume of air used in the aeration. Lythgoe (10) recommended and the length of the capillaries sealed in the upper part of the the use of a messure sufficient to measure 2 inches (50.8 mm.) of flowmeters were such that a passage of 265 liters of air per hour was possible. mercury in manometer attached to the outlet pipe. Aeration cylinder K containing the ammoniacal mixture was Pennington and Greenlee (IS) applied Folin's method to the determination of loosely bound nitrogen in meat. A small air 15 inches (38 cm.) high and 1.875 inches (4.8 cm.) in diameter. pump was used to suppl the air and the total volume of air pass- The glass tube for aeration extended to within 0.25 inch (6 mm.) ing through a battery oPfour flasks which contained the alkaline from the bottom of the cylinder. mixture was recorded on an anemometer. This method of measuring does not give the exact volume assing through each ' The flowmeters were standardized in the following manner individual aeration flask. Kober and &aves (8) ex ressed by means of the apparatus shown in Figure 2: the need for determining the exact volume of air passing tirough the ammoniacal mixture and gaged the volume of air by the time Water was allowed to flow into jar B, pinch clamp G was it takes for the air to draw a certain volume of water from one opened, the suction pump was turned on, and air was allowed to bottle to another connected by means of a glass tube. Th's, pass through the aeration apparatus. Pinchclamp H was opened. An extra outlet was introduced to however, does not measure the volume of air passing through t i e ammoniacal mixture, since the resistance offered by the aeration prevent the formation of an air space in the neck of the bell jar apparatus is so different that the volume of air passing through it to enable the bell jar to be completely filled with water, befpre in a certain time is by no means identical with the volume of the air was allowed t o displace the water contained in the Jar.
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September 15,1934
INDUSTRIAL AND ENGINEERING CHEMISTRY
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STANDARD SODIUMHYDROXIDE SOLUTION. Using a clear saturated solution of sodium hydroxide, a 0.02 N solution was prepared and standardized against Bureau of Standards acid otas sium hthalate dried a t 105' C. for 2 hours, using phenolphtffalein as inchator. STANDARD SULFURICACIDSOLUTION.A 0.02 N sulfuric acid solution was standardized against the above mentioned sodium hydroxide solution, using methyl red as indicator (0.1 per cent solution in 95 per cent alcohol).
dc
REMOVAL OF AMMONIACAL NITROGEN FROM AMMONIUM CHLORIDE SOLUTION As A FUNCTION OF VOLUMEOF AIR PASBED THROUGH ALKALINE AMMONIACAL MIXTURE. The percentage of nitrogen recovered as ammonia by aerating alkaline ammoniacal mixtures was carefully investigated first by the use of ammonium chloride solution.
V
0
FIGURE 1. AERATION APPARATUS A . Air pipe connecting aeration apparatus with air line C.
Air filter
D. Reducing valve permitting reduction of line air pressure t o that re-
quired for work E . Wash bottles containing 35 per cent sulfuric acid to remove any ammonia present in air entering apparatus H. Valves allowing further adjustment of air flow through flowmeters I . Flowmeters K. ABration cylinder L. Rubber baffles M . Spray trap N . Dispersion tube ending with bulb perforated with small holee t o allow regular distribution of air through acid solution 0. Receiver containing 10 cc. of 0.02 N sulfuric acid diluted with 50 cc. of distilled water P. Pressure regulator V . Outlet tube with spray trap
The air was permitted to escape through H , thus releasing the pressure a t F. This permitted the air to flow continuously through the apparatus. Once the bell jar was filled with water, pinchclamps G and H were closed and the timing was started by means of a stop watch. The time necessary to displace the water to the point F,corresponding to the atmospheric pressure, was noted. The difference in centimeters was read in the meantime on the flowmeter. Consecutive determinations of time flow were taken at increasing intervals of a 10 cm. difference in water level. Knowing the volume of the bell jar from the neck to point P, it was possible to determine the voIume of air passing through the a paratus in a given time and a t a given pressure as indicated on tRe flowmeters.
For each flowmeter the pressure was plotted against corresponding volume in liters of air passing per hour. the experiments described, the pressure was observed on flowmeters and the corresponding volume of air passing hour through the apparatus was read from the curves.
the In the per
MATERIAL USED STANDARD AMMONIUMCHLORIDE SOLUTION.The c. P. salt was recrystallized twice from distilled water, sublimed, and then dried a t 105' C. to constant weight. A solution of this salt corresponding to 1.5 mg. of nitrogen per 25 cc. of aqueous solution was prepared and its concentration checked by alkaline distillation into standardized sulfuric acid followed by titration. POTASSIUM CARBONATE SOLUTION.Fifty grams of otassium carbonate (anhydrous) were made up to 100 cc. wit! distilled water.
Twenty-five cubic centimeters of 0.02 N sulfuric acid were pipetted into the receiver bottle and diluted with 50 cc. of distilled water. Twenty-five cubic centimeters of standard ammonium chloride solution were pipetted into the aeration cylinder and 175 cc. of distilled water added. The cylinders were connected with the receiver bottle and the aeration apparatus. One cubic centimeter kerosene and 2 cc. potassium carbonate solution were added to the ammoniacal solution. The kerosene was added to prevent foaming. Two cubic centimeters of the potassium carbonate solution were accepted because others found it satisfactory in application to eggs.' The aeration cylinders were closed. The rubber stoppers were ficrewed down by means of metallic plates. Air was blown through the alkaline ammoniacal solution a t different speeds and for different lengths of time. The time of the experiment was noted from the moment that aeration was started until the aeration ended. Pressure was read on the flowmeters; the corresponding volume of air which passed through the ammoniacal mixture per hour was taken from the curves.
the SLction Pump
Bell jar Crystallizing dish Porcelain dish D Tra t o support bell jar G,' H . l%nchclamps I. Rubber tubing connecting a paratus with receiver bot& K . Rubber tubing connecting apparatus with suction pump A. B. C.
FIGURE2. APPARATUS FOR STANDARDIZATION OF FLOWMETERS Once the aeration was stopped, the dispersion tube and the outlft tube were carefully washed with distilled water into the receiver. The acid solutions were titrated with the standard alkali, using methyl red as the indicator. Two blanks containing 200 cc. of distilled water, 1 cc. of kerosene, and 2 cc. of potassium carbonate solution were run each time. The difference between the titration of acid solution and of blanks was taken as the measure of the ammoniacal nitrogen carried as a result of the aeration from the alkaline ammoniacal mixture ( K ,Figure 1) into the standard sulfuric acid (0,Figure 1).
In all, 119 measurements were made in duplicate at rates of aeration from 124 to 265 liters per hour in the range of 2 to 10 1 Since the apparatus contained n o provision for the removal of carbon dioxide from the air, the following experiment was performed: Eighty cubic centimeters of potassium carbonate solution were added t o 520 cc. of distilled water. The p H of this solution was found to be 11.2. Then two portions of 160 cc. each, plus I cc. of kerosene, were aerated 5 hours a t 8 rate of 240 liters of air per hour. The p H was then found t o be.lO.6. Obviously the use of the amount of potaseium carbonate specified insures an alknline reaction.
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ANALYTICAL EDITION
1
0
L
4
6
8
/O
/2
I*
0
?-;me h Hours
I
I
I
I
400
400
/ZOO
/600
I
LO00
WOO
28W
Volume o f Air in Liters
FIGURE4. AMMONIACAL NITROGENRECOVERED AS FUNCTION OF VOLUME OF AIR PASSED THROUGH ALKALINE MIXTUREAT INCREASING RATESOF AERATION
FIGURE3. AMMONIACAL NITROGENRECOVERED AS FUNCTION OF LENGTH OF TIMEAT INCREASING RATES OF AERATION hours' aeration. The data obtained in these experiments are summarized in Figures 3 and 4. The curves in Figure 3 show that the percentage of nitrogen recovered is a function of the length of time of aeration, that about half of the ammonia comes over in the first 2 hours, and that a very large volume of air is necessary to remove the last traces of ammonia. Figure 4 shows conclusively that the percentage of nitrogen recovered is primarily a function of the volume of air which passes through the ammoniacal mixture, since all the results obtained by aeration a t different speeds for increasing length of time fall on the same curve. This means that a definite volume of air, regardless of the speed of aeration in the range considered (135 to 265 liters of air per hour), will liberate a definite amount of ammonia from an ammonium chloride solution treated in the manner described. There are three main drawbacks to increasing the speed of aeration above 270 liters of air per hour with the apparatus used: At a higher speed the alkaline ammoniacal mixture may be carried over mechanically into the receiver; the acid solution may be blown out of the receiver; and the absorption of the ammonia may not be complete in the standard acid. To prevent particles from being sprayed over mechanically into the receiver, the glass tubes connecting the aeration cylinder and the receiver bottle were fitted with a glass trap. Rubber baffles were placed on the aeration tubes inside the cylinder for the same purpose. At the highest speed of aeration of 270 liters per hour as used in some experiments, controlled blank tests showed that no acid solution was blown out of the receiver. To investigate the absorption of the liberated ammonia in the receiver containing the acid solution, a second receiver, containing 10 cc. of standard sulfuric acid and 50 cc. of water, was placed in series with the first. The other conditions were the same as above. The titration of the solution in the second receiver in each experiment and of that in the blank checked within the precision of the method. Hence at a rate of aeration of 270 liters per hour the absorption of liberated ammonia is complete in the acid solution contained in the first receiver. As A FUNCTION OF VOLUMEAND CONCENTRATION OF AMMONIACAL MIXTURES. Folin and Macallum (6) called attention to the fact that the rapidity with which a given air current removes ammonia from solutions depends on the volume of the solution. The extent of the influence of volume is shown by curves I and I1 in Figure 5 . The conditions represented by the curve I experiment cannot be applied to eggs owing to the high viscosity. Use of alcohol as a diluent has been found necessary in order to inhibit foaming. Since ammonia is five times less soluble in
Vol. 6. No. 5
alcohol than in water, the ammonia should be removed more rapidly, as shown by comparison of curves I1 and 111.
REMOVAL OF LOOSELY BOUND NITROGEN FROM EGGS .The best conditions for the determination of ammoniacal nitrogen in eggs were investigated with respect to the weight of sample, total volume of alkaline egg mixture, and volume of alcohol and water added. The mixture which was found to give the least mechanical disturbances, such as splashing and sticking to the sides of the cylinder, and the order of preparation of the alkaline mixture prior to aeration were as follows: O n e g r a m of sodium fluoride was put i n t h e aeration cylinder. T w e n t y - f i v e grams of mixed egg specimen weighed by difference in a weighing bottle were put in the aeration cylinder. The contents of the cylind&r were then diluted with distilled water, the volume of added water being 75 cc. minus the number of cubic centimeters of potassium carbonate solution added later. Fifty cubic centimeters of 95 per cent ethyl alcohol 0 400 880 /Zoo /600 were added and then 1 cc. YOLUWEOFA/UIN L/TE.QS of kerosene. K e r o s e n e FIGURE 5 . INFLUENCE OF VOLand alcohol are essential UME OF AQUEOUS SOLUTION AND a s f o a m breakers. The OF SUBSTITUTION OF ALCOHOL potassium carbonate soluFOR WATERON RECOVERY OF tion was added last when AMZZONIA the whole apparatus had been connected, and the Ammonium chloride equal t o 1.5 mg. aeration was immediately of nitrogen present in each case. started. As A FUNCTIOK OF TOTAL VOLUMEOF AIR PASSED THROUGH ALKALINEEGGMIXTURE. The eggs used in these and subsequently described experiments were carefully selected at a storage and egg-breaking plant in Jersey City, N. J. Specimen I consisted of storage eggs which had been kept at 30Oto 32" F. (-1.1" to 0" C.) for 4 months. After removal from the shells, approximately 35 pounds (16 kg.) of the whole egg mixture were passed through a colloid mill to obtain a uniform magma which would not thicken on frozen storage (3). This well-mixed egg magma was poured into half-pint jars which were placed immediately in the storage room a t -5" to 0' F. (-21' to -18' C.).
Specimen I1 consisted of fresh eggs carefully selected and broken out of the shell, then mixed and stored in the same manner as specimen I. As needed, samples were delivered from Jersey City on the evening prior to their use and were stored overnight in tJhelabora-
I N D U S T R I A I, A N D E N G I N E E R I N G C H E M I S T R Y
Sept,ember 15,1934
tory at 0" to 20" F. (-18' t o -7" C.). Immediately before weighing out the samples, the frozen specimens were thawed by immersing the jars in the water of a thermostat at 25' C. During the thawing, which was completed in 20 minutes, the contents were stirred continuously.
Using specimen I with 2 cc. of the potassium carbonate solution as the alkalinizing agent, the influence of the rate of aeration a t various times was measured. The results, plotted as curve I i n Figure 6, lead to the conclusion that the recovery of loosely bound nitrogen is primarily a function of the total volume of air passed through the alkaline egg mixture and is independent of the speed of aeration in the range considered, since all the results obtained for different speeds fall on the same curve, just as was found for ammonium chloride solutions. The only advantage of increasing the velocity of aeration is shortening the time of the experiment. Inasmuch as a speed of 240 liters per hour allows complete absorption of the liberated ammonia in the 0.02 N sulfuric acid and does not cause any blowing out of the acid solution from the receiver or any mechanical carrying over of particles from the aeration cylinder to the receiving bottle, this rate of aeration was used in the subsequent experiments. Satisfactory precision was obtained, as may be seen from Table I, using egg specimen 11, aerating a t 240 liters of air per hour for 5 hours, and using 2 cc. of potassium carbonate solution containing 50 grams of potassium carbonate per 100 cc. of solution. Curve I in Figure 6 seems to indicate that 1200 liters of air are sufficient, since further aeration failed to remove more ammoniacal nitrogen. Experiments were then performed to determine whether this volume of air passing a t 240 liters per hour would be sufficient to recover the loosely bound nitrogen from egg samples containing smaller or greater amounts than specimen I.
added potassium carbonate catalyzes the hydrolysis of the egg magha with the consequence that more ammonia is split off, which is then determined as ammoniacal nitrogen. The flattening out of the curve proves that, if there is any such hydrolysis, it is not continuous.
6
i5 Ps 4 a
h
1 3
2
0, $
2
J
0 ,200
My) dm loQD /zoo NW Volume ofAir in Liters FIGURE 6. REMOVAL OF AMMONIACAL NITROGEN AS FUNCTION OF VOLUME OF AIRPASSED THROUGH ALKA-
400
LINE
MIXTURE
I. Ammoniacal nitrogen recovered from egg sample I,
ex ressed in milligrams of nitrogen per 100-gram sample. PI. Ammoniacal nitrogen recovered from standard ammonium chloride solution, expressed in milligrams of nitrogen per 100 cc. of solution.
Figure 7 gives the results obtained from different samples. Specimens I, 11, Ib, and ICwere analyzed under the same conditions, using 2 cc. of potassium carbonate solution as alkaline reagent. Specimens Ib and IC were prepared by merely keeping specimen I a t 45' F. (7" C.) for 39 and 63 hours, respectively, thus permitting slight decomposition to take place. Inspection of Figure 7 shows that additional aeration above 1200 liters failed to remove any more ammoniacal nitrogen, since the curve flattens out; the tangent to the curve a t the point corresponding to 1200 liters of aeration is practically parallel to the abscissa. This flattening out of the curve, which in the case of aeration of ammoniacal nitrogen from a standard ammonium chloride solution corresponds to 99.0 * 0.5 per cent of the actual nitrogen present, has in the case of the determination of loosely bound nitrogen in egg still another significance. It is possible that the presence of the
400
600
EW
/ooO
/ZOO
1400
VOLUME oc Am tn Ltrrrs
FIGURE7. REMOVALOF AMMONIACAL NITROGEN FROM EGGS AS FUNCTION OF VOLUME OF AIR PASSED THROUGH ALKALINE EGGMIXTURE
As .4 BONATE.
FUNCTION OF CONCENTRATION OF POTASSIUM CAR-
First the influence of different amounts of potassium carbonate upon the p H of the solutions was measured by means of the modified Bailey electrodes (2, 3). The solutions consisted of 25 grams of egg specimen 11, 1 gram of sodium fluoride, 75 minus z cc. of distilled water, and z cc. of potassium carbonate solution. Figure 8 summarizes the results. TABLE I.
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341
PRECISIOX OF
LOOSELY NUMBER FROZEN- BOUND O F E X STORAGE AGE NITROQENPERIMENTS Daws9 Ma./lOO -. -a. 7 1.34 5 8 1.41 6 9 1.37 6 10 1.25 5 11 1.14 6 14 1.05 0 16 1.32 4 4 17 1.38 21 1.34 5 24 1.33 5 2 25 1.35 26 1.31 4 27 1.34 4 30 1.35 5 31 1.35 6 36 1.38 6 48 1.37 3
MEASUREMENTS AVERAGED~VIATION Single observation b
0.04 0.02 0.02 0.04 0.04 0.04 0.02 0.02 0.04 0.02 0.00 0.01 0.01 0.01
Meane
0.02 0.01 0.01 0.02 0.02 0.02 0.01 0.01 0.02 0.01 0.00 0.006 0.006 0.005
0.02 0.01 0.01 0.005 0.01 0.008 a Number of days specimen was kept at O O F . (-18' C.) prior to analysis. b Sum of the deviations of each individual measurement from the srithmetical mean divided b the number of experiments. c Average deviation &vided by the square root of the number of exparimenta.
Then in order to measure the influence of the concentration of potassium carbonate on the liberation and removal of ammoniacal nitrogen, a series of experiments was performed which are summarized in Figure 9. From curve 0 it is obvious that an alkaline reagent must be added in order to speed up the recovery of the ammoniacal nitrogen. Curves 0.5 to 12, inclusive, show that upon increasing additions of potassium carbonate solution, increasing quantities of ammoniacal nitrogen are recovered. These curves also indicate that it is necessary to aerate the egg mixture a t an initial pH of at least 9.5 in order to obtain a quantitative recovery of ammoniacal nitrogen originally present.
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ANALYTICAL EDITION
They suggest, too, that more than 4 cc. of potassium carbonate solution should not be used to effect a definite end point for an analytically reasonable expenditure of time and effort. Curves 8 and 12 may be interpreted as showing that the corresponding amounts of potassium carbonate catalyze a hydrolytic decomposition of complex nitrogenous substances into volatile basic nitrogen compounds.
was evaluated in a standard ammonium chloride solution containing 2.99 mg. of nitrogen per 100 cc. of solution by blowing air a t a rate of 240 liters per hour, using 2 cc. of potassium carbonate solution as alkaline agent. The results obtained are plotted in curve. I1 of Figure 6. Curves I1 and I are converging; the liberation of ammoniacal nitrogen in eggs is similar to that of standard ammonium salt solutions, when the percentage of ammoniacal nitrogen is approximately the same and when the conditions of the experiment are the same. This investigation shows, however, that when the conditions of rate and time of aeration, pH, and volume of the egg mixture are controlled, it is unnecessary to run ammonium salt controls. DETAILSOF METHOD As a result of this investigation, the authors recommend the following method for the precise determination of the loosely bound nitrogen in eggs, using the apparatus described in Figure 1:
FIGURE8. PH OF AQUEOUS EGGMIXTURE, AS FUNCTIONOF AMOUNTOF POTASSIUM CARBONATE SOLUTIONADDED
It is known that increasing alkalinity as effected by use of sodium hydroxide yields increasing amounts of ammoniacal nitrogen from eggs (6). Also, increasing amounts of sodium carbonate producing pH values over 9.0 have been shown to cause decomposition of amino acids a t about 100" C. ( 1 2 ) . Results such as those represented by curves 0.5, 2, and 4 in Figure 9 have not been heretofore demonstrated. The selection of any of these three conditions is indicated as proper for the purpose and consequently it is well to adhere to the previous empirical selection of potassium carbonate solution, giving an initial p H of about 10.3. VALIDITY AND USE OF STANDARD AMMONIA CONTROLS
It has been customary with some workers to run parallel aerations of a n ammonium chloride or ammonium sulfate solution to serve as a gage of the efficiency of the analysis of an egg specimen. If over 95 per cent of the actual amount of nitrogen present in the solution was recovered, it was concluded that approximately the same percentage of ammoniacal nitrogen present in the egg sample had been recovered. However, such a conclusion is possible only when the per-
Measure accurately 10 cc. of 0.02 N sulfuric acid into the receiver, 0, and dilute with 50 cc. of distilled water. Place 1 gram of sodium fluoride in aeration cylinder K. Pour 25 grams of mixed egg magma, weighed by difference from a weighing bottle, into the aeration cylinder. Add 55 cc. of distilled water, 50 cc. of ethyl alcohol (95 per cent by volume), and 1 cc. of kerosene. Connect the cylinders with the receiver bottle and the aeration apparatus. Add 20 cc. of potassium carbonate solution, containing 5 grams of anhydrous potassium carbonate cc. of solution. Close the aeration cylinders, and start tE?a6%! tion, allowing the passage of 1200 liters of air at a rate of 240 liters per hour. Titrate the excess of acid with 0.02 N sodium hydroxide (free from carbon dioxide) by means of a microburet which can be read t o 0.01 cc. Run at the same time two blank experiments, containing 1 gram of sodium fluoride, 80 cc. of distilled water, 50 cc. of 95 per cent alcohol, 1 cc. of kerosene, and 20 cc. of potassium carbonate s'olution. Take the difference between the titration of excess acid and of blanks as the measure of the ammoniacal nitrogen carried as a result of the aeration fr m the alkaline egg mixture, K , into the standard sulfuric acid, Express the results obtained as milligrams of l'ammonia nitrogen" per 100 grams of sample.
8.
From a consideration of all the errors involved in the above method, the precision is indicated to be j=2 per cent. ACKNOWLEDGMENT The authors wish to express their indebtedness to Samuel B. Ellis for his cooperation in the construction and standardization of the flowmeters used. LITERATURE CITED
Alfend, S., J. Assoc. O$ciaZ Aor. Chem., 15, 336 (1932). Bailey, C. H., J. Am. Chem. Soc., 42, 45 (1920). Bailey, M. I., Dissertation, Columbia University, 1932; Thomas, A. W., and Bailey, M. I., IND.ENG.C H ~ M25, ., 669 (1933). Folin, O., 2.' physiol. Chem., 37, 161 (1902). Folin, O., and Macallum, A. B., J. BioZ. Chem., 11, 532 (1912). Hendrickson, N., and Swan, G. C., IND. ENQ.CHEM.,10,614 (1918). Hertwig, R., J. Assoc. Oflcial Agr. Chem., 8, 594 (1925'1. Kober, P. A., and Graves, S. S., J. Am. Chem. Soc., 35, 1594 (1913). 250 500 750 IO00 /ZSO IS00 /7SO Lourie, H. L., J . Assoc. Oficial Agr. Chem., 6 , 4 (1922). Vo/ume of Air in Liters Lythgoe, H. C., IND. ENQ.CHEM.,19, 922 (1927). FIGURE9. REMOVALOF AMMONIACAL NITROGEN FROM EGG Marsh, C. E., J. Assoc. Oflcial Agr. Chem., 4, 507 (1921). MIXTURESCONTAININGVARYINGAMOUNTSOF POTASSIUM Nichols, H. S.,and Foote, M. F., IND.ENG.CHEM., Anal. Ed., CARBONATE 3, 311 (1931). (13) . . Penninaton, M. E., and Greenlee, A. D., J.Am. Chem. Soc., 32, 561 (1910). centage of the ammoniacal nitrogen is approximately the (14) Redfield, H. W., J. A ~ s O C . Oficial 4 7 . Chem.,4, 516 (1921). same in both egg mixture and ammonium &loride solution (15) Redfield, H. W., U. S. Dept. apric., Bur. Chem., Bull. 846 and when all other conditions are identical. (1920).
Since egg specimen I contained approximately 3 mg. of ammoniacal nitrogen per 100 grams, the ammoniacal nitrogen
RE)CIIVB)D October 2. 1933.
