Microdetermination of Ammonia Nitrogen in Eggs SELMA L. BANDEMER AND PHILIP J. SCHAIBLE Michigan Agricultural Experiment Station, East Lansing, Mich.
I
NTERIOR quality of shell
surface and when used were A method for the determination of ameggs is commonly and most coated with a thin film of vasemonia nitrogen in eggs without aeration is line to insure a gas-tight seal. conveniently determined in comdescribed. The ammonia is absorbed in By slipping the cover horizonmerce upon the unopened egg standard acid from a thin layer of an alkatally until there was an opening by means of candling. Physiline solution of the sample in a modificajust large enough for the tip of cal methods that measure quality a pipet, solutions were added to more precisely have been detion of the Conway-Byrne cell. I t has the cell without either absorpscribed for eggs which may be many advantages over the troublesome tion of ammonia from the atbroken out (3, 4). Chemical aeration methods in that larger numbers of mosphere or loss of ammonia methods have not found applicasamples may be run simultaneously and from the cell. Figure 1 shows tion in this field except for comthe operation, control, and equipment are the cell and indicates the method paratively few studies of the of introduction of materials. In loosely bound nitrogen (2, 5 ) . simpler. Data are presented to show the washing these cells i t was found The chemical procedure hitherto reliability and accuracy of the procedure. advisable to rinse them in a 1 to followed has been an adaptation Directions are given for the fabrication of 1 hydrochloric acid solution to of the Folin aeration method for the modified Conway-Byrne cell. remove the last traces of the a m m o n i a i n urine-that is, alkali. eweciallv from the inner aeration of an alkaline solution of the material, absorption of the ammonia by excess standard chamber. Ostwald-Folin pipets Jiere'used io sample the egg malerial. Tips were adjusted tjo allow rapid delivery and acid, and its subsequent determination by titration or nesslerithey were then calibrated to deliver 5 cc. of egg white, yolk, zation. The aeration method has many disadvantages, especially diluted yolk, or mixed whole egg. An electrically heated when applied to the estimation of the extremely small amounts cabinet kept a t 38" * 1' C. by means of a thermostat was used to heat the cells for the required time. of ammonia in egg materials. It involves a complicated setup of wash bottles, aeration tubes, absorption tubes, maSolutions Required nometers, pressure regulators, and traps, since the volume of The acid used was 0.005 N hydrochloric acid, made by air, its rate of flow, and its pressure must be carefully regulated and its freedom from ammonia insured (2, 5 ) . Excesdiluting 0.10 N hydrochloric acid, and contained the indicator. sive foaming must be prevented, which is very difficult with The indicator, adapted from that described by Tashiro and modified by Conway ( I ) , was prepared by dissolving 0.08 egg materials. The time involved for aeration is between 4 and 5 hours, and the attention of the operator is required gram of the sodium salt of methyl red and 0.02 gram of methylene blue separately in alcohol, mixing, and making up during this period. to 100 cc. wit8halcohol. One cubic centimeter of this solution To overcome the inconvenience of the aeration method an attempt was made to use the absorption process described by was added to each 100 cc. of the standard acid before making Conway and Byrne ( I ) , but it was found necessary to modify to volume. With this indicator the end point was that point t h i s p r o c e d u r e to adapt it to a t which t h e least color was the purpose a t hand. The prinpresent, t h e s o l u t i o n h a v i n g a grayish tinge between the red of ciple involved is the l i b e r a t i o n the acid and the green of the alkaof ammonia by a saturated poline colors. The end point was tassium carbonate s o l u t i o n and very sharp and readily reproducible, its absorption in standard acid as titrations checking within 0.01 cc. before, but instead of its removal by aeration, the volatility of the The excess acid was determined by ammonia is employed for this purtitration with 0.0025 N sodium hydroxide, also prepared by diluting a pose. 0.1 N solution and standardized Apparatus each d a y a g a i n s t the acid. A Cells were made f r o m t o p s of saturated potassium carbonate solu10-cm. Petri dishes. The rims of tion was used to liberate the amt h e s e d i s h e s were ground to a monia from the egg m i x t u r e s . Ammonia-free water prepared by smooth surface on a piece of plate glass with the aid of fine CarborunFIGURE1. A B S O R P T I OCELL, N I N D I C A T I N G distilling distilled water containing OF INTRODUCING MATERIALS METHOD dum powder and water. Glass a small amount of potassium acid rings 5 cm. in diameter and of a sulfate was used throughout, and it height slightly less than that of the side walls were centered in was found advisable to carry out the determinations in an the dishes and sealed in place with de Khotinsky cement, which ammonia-free atmosphere. Suitable blanks were run. in turn was coated with beeswax to protect the cement from Method the alkali used. This wax coating had Bo be renewed a t intervals. An all-glass cell proved very satisfactory but was This method was used for whites and yolks separately and unduly expensive. Covers were glass plates with a ground for mixed whole eggs. The eggs to be examined were 201
202
IKDUSTRIAL AND ENGINEERING CHEMISTRY
broken out into Petri dishes and samples taken as described below. EQQWHITE. Place in the inner chamber 5 cc. of 0.006 N hydrochloric acid from a calibrated pipet and in the outer chamber approximately 2 cc+of ammonia-free water. Cover the cell. Slip the cover horizontally slightly to provide a narrow opening and add t o the outer chamber 5 cc. of the liquid white from the pipet calibrated for this purpose. Close the cell and mix the contents by gently tilting and rotating. Introduce approximately 2 CC. of saturated potassium carbonate solution into the outer chamber and again mix the contents as before. Place the cells in stacks with a kilogram weight on topin the cabinet kept at 38" C. for 1.5 hours. At the end of this period, titrate the excess acid with 0.0025 N sodium hydroxide solution, and calculate the ammonia to milligrams of ammonia nitrogen per 100 cc. of egg white.
EGGYOLK. This procedure has been slightly modified by a preliminary dilution of yolk, since undiluted yolk does not mix properly with the carbonate solution under the conditions specified for egg white, After removing the sample of white, slit the yolk membrane slightly with a small scalpel at the top of the olk without removing the firm white. Thrust the Ostwald-Fogn pipet with an enlarged tip through the opening, remove 5 cc. of yolk, and add to 3 cc. of ammonia-free water in a test tube. The yolk can be conveniently blown out by means of a rubber bulb attached to the pipet. After thorou hly mixing, introduce a 5-cc. aliquot with the pipet calibrated i%r diluted yolk into the outer chamber of a cell containing 5 cc. of the standard acid in the inner chamber. Add 3 cc. of the saturated potassium carbonate solution to the diluted yolk, close the cell, mix with the same rotary and tilting motion, and place in the cabinet with a weight for 1.5 hours at 38" C. As before, titrate the excess acid with 0.0025 N sodium hydroxide and calculate the ammonia to milligrams of ammonia nitrogen per 100 cc. of yolk.
MIXED WHOLEEGG. Mixed whole egg was prepared by breaking an egg in a 6-ounce wide-mouth glass-stoppered bottle and shaking it with four large glass beads. The procedure then used was practically the same as for egg yolk. Deliver 5 cc. of mixed whole egg with the Ostwald-Folin pipet calibrated for mixed whole egg into the outer chamber of a cell which contains 5 cc. of the standard acid in the inner chamber. Add 3 cc. of the saturated potassium carbonate solution, close the cell, mix by tilting and rotating, and place in the cabinet for 1.5 hours at 38" C. Titrate the excess acid and calculate in terms of milligrams of ammonia nitrogen per 100 cc. whole egg. Experimental Conway and Byrne (I) have suggested alternative sets of conditions for the quantitative absorption of ammonia, stipulating 1.5 hours a t room temperature or 1 hour a t 38" C. The ammonia nitrogen is so low in fresh eggs that larger samples are required than can satisfactorily be handled in the Conway-Byrne unit. With the modification of the unit suggested in this paper the interrelationships of time, temperature, and linear dimensions of the inner and outer chambers require a longer period or a higher temperature than that specified by Conway and Byrne. In practice, holding periods at room temperature up to 3 hours or a t 38" C. less than 1.5 hours did not provide suitable conditions for recovery of ammonia from egg materials. As a result a period of 1.5 hours a t 38" C. was chosen. The volume of saturated solution of potassium carbonate required was determined by experiment with white and yolk, keeping other factors constant. From Table I it was evident that the quantity of saturated solution of potassium carbonate could be varied considerably without changing the amount of ammonia liberated. Since it was desirable to keep the volume in the outer chamber a8 small as possible and there was no advantage in using larger volumes, it was deemed sufficientto use for the white 2 00. of the saturated solution of potassium carbonate and for the yolk, 3 cc.
VOL. 8, NO. 3
TABLE I. SATURATED SOLUTION O F POTASSIUM CARBONATE REQUIRED FOR COMPLETE EXPULSION OF AMMONIA ICZCOI Solution Used
cc.
Ammonia Nitrogen White Yolk M g . / i O O ec.
lVQ./100 CC.
2.30 2.63 2.60 2.58 2.63
1 2 3
0:is 0.52 0.45
5
0.46
4
The possibility that ammonia might be liberated from yolk alone during the period of heating was investigated and the results are given in Table 11. TABLE11. EFFECTOF HOLDING YOLKAT 38" C. FOR 1.5 HOURS WITHOUT ADDITIONOF CARBONATE SOLUTION 0.005 N Alkali Required to Titrate Acid
cc. NHa-free water "a-free
water
4.52 4.54 4.58 4.54 4.54 4.52
+ yolk
Since titration values of the standard acid were the same with and without the yolk, it is certain that no ammonia was liberated by the yolk itself when held for the required time without the carbonate additmion. This is probably due to the fact that yolk is naturally slightly acid, having a pH of approximately 6.0 when fresh which increases to about pH 6.8 after long periods of storage. The absorption period was established as the time required for the recovery of ammonia from a standard ammonium sulfate solution a t 38" C. TABLE111. TIMENECESSARY FOR COMPLETE REMOVAL OF AMMONIUM NITROGEN FROM AMMONIUM SULFATE SOLUTION (Results expreseed as milligrams of ammonia nitrogen in 100 cc. of Bolution.) Concentration of Solution Concentration of Solution Used by Actual Determination Time a t 38" C. Hours 4.11 4.11 4.11 4.11
0.5 1.0 1.5 2.0
2.45 3.56
4.07
4.03
Since an absorption period of 1.5 hours gave practically the theoretical value for the ammonia nitrogen recovered, this time was used for all the experiments. The recovery of ammonia from a standard ammonium sulfate solution replacing yolk or when added to yolk was found to be satisfactory. Varying quantities of a standard ammonium sulfate solution were used in a series of determinations to produce concentrations similar to what might be expected in egg yolk. O F AMMONIANITROQEN FROM A TABLEIv. RECOVERY STANDARD SOLUTION OF AMMONIUM SULFATE
No. of Detns. 7 10 7 7
Ammonia Nitrogen Recovered Added Mg./100 CC. Mg./100 eo. 2.05 4.11 5.00 6.16
2.04 4.06 5.24
5.97
Deviation Standard
0.143 0.145 0.080 0.365
A comparison of the amounts added and recovered in Table IV shows satisfactory agreement. When definite amounts of ammonia nitrogen were added to yolk and determinations made before and after the additions, the results shown in Table V were obtained.
MAY 15, 1936
-4NALYTICAL E D I T I O N
In Table V three different quantities of ammonia nitrogen were added to aliquots of two lots of pooled yolk. The recovery of the ammonia nitrogen was deemed satisfactory in all cases. The period of 1.5 hours a t 38" C. was used for the experiments reported in Tables IV and V and for results reported below.
v.
RECOVERY O F AMMONIA NITROGEN FROM EGG TABLE YOLK CONTA41NING VARYING ADDITIONSOF AMMONIUM
Estimation of Oleomargarine in Ice Cream T. H. W-HITEHEAD AND J. B. DUNSON University of Georgia, Athens, Ga.
SULFATE
(Results expressed as milligrams of ammonia nitrogen per 100 cc.) No. of Determined on Added Calculated .4ctually Detns. Yolk Alone t o Yolk Total Determined 5.28 5.23 4 3.47 1.81 4.30 4.40 4 2.49 1.81 6.85 6.76 4 3.47 3.38 5,87 5.80 4 2.49 3.38 4 3.47 4.73 8.20 8.23 7.22 7.39 4 2.49 4.73
In the procedure for egg white described above, the directions specify the use of liquid white. Firm white could also be used, but the liquid white is much easier to pipet and mix with the carbonate solution than the firm white and has the same percentage of ammonia nitrogen for the same egg. This is ~ h o w nin Table VI, where the values for the three distinct layers of egg white for four eggs are given. All the results are within experimental error. In practice it was found satisfactory to slit the firm white envelope and pipet from the mixture of outside and inside liquid white. TABLE VI. DISTRIBUTION OF AMMONIANITROGEN IN Eaa WHITF. (Results expressed as milligrams of ammonia nitrogen per 100 cc.) Outside .Liquid Inside Liquid Egg White Firm White White 1 0.12 0.10 0 11 2 0 14 0.18 0 21 0 18 0.12 3 0.12 4 0 20 0 21 0 20
In a large number of eggs of similar history the ammonia nitrogen was determined for both white and yolk by the aeration and absorption methods. Both methods were not applied on the same samples. Reference to Table VI1 shows that in general there was satisfactory agreement between the values obtained by the two methods for both white and yolk on material of similar nature. O F THE ANALYSIS TAElLE VII. COMPARISON OF RESULTS OF EGGS BY AERATION AND ABSORPTION METHODS
Method
Material
&44)ration
White Yolk White Yolk .Mixed whole egg
Absorption
No. of Ammonia Standard Samples Nitrogen Deviation Mg./IOO cc. 83 0.24 0.086 83 2.30 0.287 16 0.35 0.137 301 2.41 0.280 6 0.87 0.003
Acknowledgment The eggs used in this study were kindly supplied by the Poultry Section of the Experiment Station.
Literature Cited (1) Conway, E. J., and Byme, A., Biochem. J.,2 7 , 4 1 9 (1933). (2) Hendrickson, N., and Swan,G. C., J. IND. ENG.CHEM.,10, 6 1 4 (1918). (3) Holst, W. F., and Slmquist, H. J., Hzlgurdiu, 6 , 4 9 (1931). (4) Sharp, P. F., and Powell, C. K., IND.ENO.CHEM.,22, 905 (1930). (5) Thomas, -4.W., and Van Hauwaert, M. A., Zbid., Anal. Ed., 6 , 338 (1934). RECEIVE^) February 8 , 1936. Published with the permission of the Director of the Experiment Station as Journal Article 251 (n. a,).
203
A method for determining the percentage of oleomargarine and butter fat in ice cream is given, which consists of a modified Roese-Gottlieb extraction procedure combined with a modified Reichert-Meissl procedure. Formulas are derived for calculating the percentage of oleomargarine in the extracted fat, the fat being calculated in the usual manner.
T
HE lower price of ole-margarine as compared to butter fat a t present has led some manufacturers of ice cream to substitute oleomargarine to some degree for butter fat. This may soon bring about serious consequences; it is illegal in many states and some investigators (3) have shown that ice cream made of olecmargarine is deficient in certain vitamins. It would seem obviously unfair to sell as ice cream a product that is not made of butter fat, but any ruling of fair trade competition must first have some basis for classification. This problem led the authors to work on a method for determining the relative amounts of oleomargarine and butter fat in ice cream with a reasonable degree of accuracy. The problem is more difficult than it first seemed, Modern homogenizing equipment has made microscopic examination and staining almost useless as a means of detecting oleomargarine, and refractive index and specific gravity gave no suitable quantitative data. The iodine value gave qualitative indication, but because of the diversity of oleomargarines on the market did not give quantitative confirmation. The Kirschner-Flanders (4)method did not give quantitative results when tried on the authors' samples. The usual Reichert-Meissl method (1)was also soniewhat unsatisfactory and gave erratic results. However, it was found that if the Roese-Gottlieb extraction procedure and the Reichert-Meissl procedure were slightly modified, a precise method was obtained. Experimental
APPARATUS. The usual apparatus for determining the fat percentage in butter was used (6). REAGENTS. Oleomargarine was furnished by the Joseph Costa Co., Athens Ga., and had the following composition: 70 Total fat Oleic oil Neutral oil Cottonseed oil Moisture Salt Milk solids
34 34
15
% 83
12 3 2
Ammonium hydroxide, ethyl alcohol, diethyl ether, and petroleum ether as given by the method of the Association of Official Agricultural Chemist (2). Sulfuric acid: 31.5 cc. of acid (sp. gr. 1.83) per liter.
