Determination of Uric Acid in the Mixed Excrements of Birds A Modification of the Fritz Differential Extraction Method RAY L. SHIRLEY AND A. H. VANLANDINGHAM West Virginia Agricultural Experiment Station, Morgantown, W. Va.
F
RITZ (1) has discussed the need for an accurate determination of the uric acid in the mixed excrements of birds for the calculation of the digestion coefficient of proteins. He subjected several methods for the determination of uric acid to critical test and was unable to obtain satisfactory results with any of them. He then proposed a new differential extraction method and recommended it because of its simplicity and accuracy. The differential extraction method is based on the assumption that acidified water extracts the same amount of nitrogen, with the exception of uric acid nitrogen, as does piperidine. Two equal samples of the excrement (2-gram samples are recommended as a convenient quantity t o handle) are treated with 20 ml. of dilute hydrochloric acid (5 parts of concentrated hydrochloric acid and 95 parts of water) and allowed to stand overnight. The samples are filtered, the residues are washed with about 25 ml. of cold water, and the residues and filter papers are transferred to the original beakers. To one of the residues are added about 25 ml. of water and sufficient piperidine t o make the sample distinctly alkaline t o phenolphthalein. To the other residue are added 25 ml. of 0.1 N hydrochloric acid. Both samples are digested in a water bath at 60" C. for 1 hour, and the extracts are filtered off through a layer of Celite on a filter cloth. The residues are washed with equal quantities of cold water until the wash water from the piperidine-extracted material is free from an alkaline reaction. The residues and Celite are transferred to Kjeldahl flasks and total nitrogen is determined. The difference in the nitrogen content of the acid- and piperidine-extracted residues represents the uric acid nitrogen in the sample.
ing amounts of uric acid when added to chicken excrement is presented in Table I. With the modifications used Table I shows that fairly satisfactory results were obtained in the recovery of uric acid added to chicken excrement. However, this is not necessarily evidence that equivalent amounts of nonuric acid nitrogen are extracted from chicken excrement by acidified water and piperidine, as is assumed in the development of the method. TABLE11. APPARENTVALUESFOR URIC ACID os URICACIDFREEMATERI.4L BY THE FRITZ METHOD Intestinal Contents of Hens
Sample
Lower half
1
Upper half
Nitrogen in Residues Extracted with: 0.1 N HC1 Piperidine
Uric Acid Nitrogen
%
%
%
Uric Acid %
0.49 0.49 0.490
0.26
0.78
2 Av.
0.75 0.76 0.755
0.27 0.266
0.81 0.795
1 2 Av.
0.74
0.74 0.740
0.37 0.36 0.365
0.37 0.38 0.376
1.14 1.126
1.11
In an effort to verify this assumption some determinations were made on cow feces, which mere found to contain no uric acid when an alkaline extract was tested qualitatively with arsenophosphotungstic acid and sodium cyanide. The residue from the acid extraction contained 1.565 per cent of nitrogen whereas the residue from the piperidine extraction contained 1.325 per cent of nitrogen, thus giving a difference of 0.24 per cent of nitrogen which would be considered as uric TABLE I. RECOVERY OF URIC ACID ADDED TO A STAXDARD acid nitrogen by this method. I n order to obtain further eviOF CHICKEN EXCREMENT SAMPLE dence on this point seven young hens were confined in cages Uric Acid Uric Acid Recovery of Added and a composite sample of excrement was collected. The hens Sample Added Found Uric Acid were then slaughtered and the contents of two different parts of Cram Gram % the small intestine were removed as separate portions. The 0.1017 101.7 0.1000 0.0756 100.8 0.0750 first portion was removed from the lower half and the second 0.0513 102.6 0.0500 0,0258 103.2 0.0250 portion from the upper half of the small intestine. The whole 0.0100 0.0114 114.0 excrement and the different portions of the contents of the 0.0108 72.0 0.0150 0.0100 0,0099 99.0 small intestine were acidified with hydrochloric acid and pre0.0100 0.0093 93.0 pared for analysis. Av. 98.3 The material removed from the small intestine of the hens was shown to be free of uric acid when tested colorimetrically The method as outlined by Fritz was tried on a number of with arsenophosphotungstic acid and sodium cyanide. Howsamples of chicken excrement collected and prepared accordever, when this material was subjected to analysis by the ing to the method of St. John and Johnson ( 2 ) with fairly differential extraction method, using 0.1 N hydrochloric acid satisfactory results. However, with some of the samples and piperidine, the results shown in Table I1 mere obtained. difficulty was encountered in obtaining satisfactory dupliIt is evident from the data presented in Table I1 that 0.1 S cate determinations for nitrogen on the piperidine-extracted hydrochloric acid and piperidine do not extract equivalent residues. This difficulty was probably due t o improper washamounts of nitrogen from materials similar to chicken excreing of the alkaline piperidine-extracted residues, the washing ment that were shown to be free of uric acid by qualitative process being rather tedious and time-consuming. tests. In a study of a number of reagents in which uric acid is Experimental soluble, it was found that diethanolamine came nearest to 0.1 N hydrochloric acid in extracting nonuric, acid nitrogen In an attempt to improve on the method of filtration and from cow feces, and a t the same time it extracted added uric washing of the piperidine-extracted residues, it was found acid quantitatively. When the concentration of diethanolthat using 1-gram samples instead of the recommended 2amine was increased to approximately three times the amount gram samples and substituting centrifugation for filtration required to make the sample distinctly alkaline to phenoleffected considerable saving of time and increased accuracy in phthalein, there was no reduction in the percentage of nitrogen the determination. With these changes, the recovery of vary381
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INDUSTRIAL AND ENGINEERING CHEMISTRY
in the extracted residue. However, when the concentration
of hydrochloric acid was increased from 0.1 to 0.5, 1.0, and 2.0 N , respectively, there was a continuous small decrease in
VOL. 11, NO. 7
tracted from the corresponding value for the residues extracted with hydrochloric acid is equal to 1.93 per cent of uric acid nitrogen or 5.79 per cent of uric acid.
the percentage of nitrogen in the acid-extracted residues.
N hydrochloric acid extracted almost identically the same amount of nitrogen from the sample as diethanolamine. Time and temperature of digestion and number of washings were varied on both the diethanolamine and hydrochloric acid extractions. Decreasing the time of digestion from 1 hour to 40-, 15-, lo-, and 5-minute periods a t 60" C. had no effect on the percentage of residue nitrogen following either the hydrochloric acid or the diethanolamine extractions. Decreasing the temperature of digestion from 60' C. to SOo, 40°,and room temperature (28" C.) for 1 hour caused a continuous slight increase in residual nitrogen after extraction with hydrochloric acid or diethanolamine. When the number of washings was varied very significant results were obtained. With the diethanolamine extraction each washing up t o and including the third showed a decided decrease in percentage of residue nitrogen, but further washing was without effect. The wash water was also free of alkalinity to phenolphthalein at the end Of the third washing* The residual nitrogen following the acid extraction was constant after the first washing. TABLE111. URICACID VALUES (Using N hydrochloric acid and diethanolamine as extractive reagents for fecal material) Nitrogen in Residues Extracted with: Uric DiethanolAcid Uric Material Sample N HC1 amine Nitrogen Acid Cow feces
Poultry feces Lower half of intestinal contents Upper half of intestinal contents Normal excrement
%
%
%
1.57 1.57
1.65
1.510
1.56 1.650
0.02 0.02 0.020
0.06 0.06 0.060
1 2 Av.
0.76 0.76 0.760
0.79 0.77 0.780
-0 01 -0.03 -0,020
-0.03 -0.09 -0.060
1 2 Bv. 1 2 Av.
0.83 0.84 0.835 3.43 3.45 3.440
0.80 0.81 0.805 0.86 0.84 0.850
0.03 0.03 0.030 2 57 2.61 2.590
0.09 0.09 0.090 7.71 7.83 7.770
1 2 Av.
%
As a result of these experiments diethanolamine and N hydrochloric acid were substituted for piperidine and 0.1 N hydrochloric acid, respectively; time of digestion a t 60 O C. was decreased from 1hour to 10 minutes; the diethanolamineextracted residue was washed three times with distilled water and the acid-extracted residue was washed once with distilled water, instead of washing both residues until the diethanolamine-extracted residue was free of alkalinity to phenolphthalein. With these changes the method was applied to uric acid-free cow feces, to material taken from the small intestines of young hens, and to whole excrement obtained before €he hens were killed, with the results shown in Table 111. These data show that N hydrochloric acid and diethanolamine extract equivalent amounts of nonuric acid nitrogen from materials similar to chicken excrement free of uric acid. This is taken as evidence that the difference in the residual nitrogen of chicken excrement samples following extraction with N hydrochloric acid and diethanolamine represents the uric acid nitrogen present. As a result of 15 determinations of uric acid on a standard sample of chicken excrement, the residues following extraction with N hydrochloric acid contained from 2.98 to 3.07 with an average of 3.03 per cent of nitrogen. The residues following the diethanolamine extractions contained from 1.09 to 1.11 with an average of 1.10 per cent of nitrogen. The nitrogen value for $he diethanolamine-extracted residues sub-
TABLE IV. RECOVERY OF ADDEDURICACID BY NEWMODIFIED PROCEDURE sample 1
2 3 4 5
6 7 8
9
10 11
12 13 14
Uric Acid -4dded Gram 0,0600
0.0900 0.0450 0.0960 0.0726 0.0521 0.1092 0.0723 0.1043 0.0524 0.0100 0.0100 0.0108 0.0103
Added Uric Acid Found Gram 0.0501 0.0900 0.0432 0.0954 0.0705 0.0513 0.1101 0.0723 0.1044 0.0510 0.0096 0.0090 0.0099 0.0108
Recovery of Added Uric Acid
% 100.2 100.0 96.0 100.4 96.2 98.5 100.8 100.0 100.1 97.3 96.0 90.0 91.7 104.8 Av. 98.0
I n Table IV are presented data showing recovery of varying
amountsof uric acid when added to the standard sample of chicken excrement. Table IV shows the general efficiency of the modified differential extraction method in the recovery of uric acid added to samples of poultry excrement. The poorer individual recoveries may be accounted for to some extent by the smaller amounts of uric acid added in these cases. The general average of 98 per cent for all recoveries is 4.2 per cent higher than that reported by Fritz for his method.
Recommended Procedure The chicken excrement is collected and mixed with approximately 1 ml. of 0.45 N hydrochloric acid per gram. The acid converts urates t o free uric acid and prevents loss of nitrogen present as ammonia. The excess moisture is driven off on a water bath and the sample is then dried to constant weight in an air oven at 100" C . The sample is ground in a Wiley mill to pass a 1-mm. sieve and allowed to come to air-dry condition before analysis. Two 1-gram samples are weighed out and transferred to 100-ml. centrifuge tubes. To one are added about 15 ml. of water and approximately twice as much 10 per cent diethanolamine as is necessary to make the mixture alkaline to phenolphthalein (6 to 8 ml. are usually sufficient) and it is then diluted to 25 ml.; to the other are added 25 ml. of N hydrochloric acid. Both s2m les are digested with frequent stirring in a water bath at 60 $for 10 minutes. The samples are removed from the water bath and allowed to cool t o room temperature, then thoroughly mixed with the aid of a rubber policeman, and the walls of the tubes washed down with a few milliliters of water. The tubes are centrifuged at about 1500 r. p. m. for about 5 minutes. The supernatant liquid is poured off and the tubes are allowed to drain for a few minutes. The diethanolamine-extracted sample is washed three times, or until the wash water is no longer alkaline to phenolphthalein, with 50 to 70 ml. of distilled water for each wash and centrifuged as before. The hydrochloric acid-extracted sample is washed only once with 50 to 70 ml. of water. After each centrifugation the residues are thoroughly broken up with a stirring rod to ensure efficient and thorough washing. After washing, the residues are transferred t o Kjeldahl flasks for total nitrogen determinations. The difference in nitrogen between the N hydrochloric acid-extracted residue and the diethanolamineextracted residue represents the uric acid nitrogen in the sample. Uric acid nitrogen X 3
=
uric acid
Summary A critical study was made of the differential extraction method for the determination of uric acid in chicken excrement. The method was modified to use 1-gram samples in place of 2-gram samples, and centrifugation in place of filtration. These changes resulted in a considerable saving of time and
JULY 15, 1939
383
ANALYTICAL EDITION
a greater accuracy for the determination. N hydrochloric acid and diethanolamine are recommended as the extractive reagents, since it was shown that 0.1 N hydrochloric acid and piperidine do not extract equivalent amounts of nonuric acid nitrogen from materials similar to chicken excrement free of uric acid.
Acknowledgment The authors wish to thank I. J. Duncan of the Depart-
ment of Agricultural Chemistry for valuable suggestions made in connection with this investigation.
Literature Cited (1) Fritz, J. C., IND.ENG.CHEM.,Anal. Ed., 7, 123 (1935). (2) St. John, J. L., and Johnson, O., J. Biol. Chern., 92, 41 (1931). P R E S ~ N Tbefore E D the Division of Biological Chemistry a t the 97th Meeting of the American Chemical Society, Baltimore, Md. Published with the approval of the Director of the West Virginia Agricultural Experiment Station as Scientifio Paper No. 218.
Determination of Electrometric Equivalence Points JOHN R. GAY Geological Survey, Washington, D. C.
T
HE staff of Research Project No. 4 of the American Pe-
troleum Institute, in their study (9) of organic matter included in sedimentary rocks, employed a modification of the analytical method of Schollenberger (8). This method of analysis involves the oxidation of the powdered sediment with a predetermined amount of 0.4 N chromic acid, in excess, and the titration of the unreduced acid with 0.2 N ferrous ammonium sulfate solution. The end point in this titration has been determined with the aid of an internal indicator, diphenylamine.
R
rnkchanicol stirrer
FIGURE 1. SCHEMATIC-PICTORIAL DIAGRAM OF TITRIMETER a, b. Poles for two circuits controlled b y switch S
E.
Cell composed of electrodes and electrolyte
F. Dry cell (1.5 volts)
M . Milliammeter, 0 t o 1 milliampere, 1000 ohms per volt sensitivity (Triplett model 221 or 321) Pt. Platinum electrode 22. 1500-ohm, 1-watt, rheostat-type control (wire wound) 5. Single-pole double-throw knife switch W. Tungsten electrode
However, difficulty is encountered in detecting the end point of this particular titration. The suspended particles in the titration vessel may mask or even prevent the observation of the color change, especially with iron-rich sediments. The color of the trivalent chromium is an intense blue, affecting the normal purple hue of the oxidized indicator. More-
over, the indicator in the oxidized condition is a dye, which decomposes gradually and forms a dense, black, tarry deposit around the edges of the solution in the beaker; also, a strong source of light is needed in order to observe the gradual change of the color of the indicator. The indicator has to be added carefully, and a beginner usually requires several days of experience before he becomes adept in detecting the endpoint color changes. These disadvantages are eliminated by electrometric titration. Present methods of detecting equivalence points resolve themselves into two main groups. The first and oldest method involves the use of a sensitive potentiometer. The practice is to balance the potential of cell E (Figure 1) with a known potential, and, in this way, to determine the greatest potential change for the smallest volume of titrant added (1). It is necessary to plot the potential changes throughout the entire reaction, in order to ascertain the equivalence point. The second method employs the vacuum-tube voltmeter, and is a comparatively recent development. In this method, the cell potential is measured directly, without drawing any current from the cell. The cell potential is impressed in static charge on the control grid of the vacuum tube, and has a control or Thyratron action on the flow of plate current of the tube. Variations in the plate current are either detected in the first stage or amplified further by a cascade amplifier. The main point is that in both methods no current is drawn from the cell, so that polarization of the cell is prevented. Polarization causes the formation of gases on both of the electrodes, and halts the action of the cell. The potentiometric method involves continual manipulation throughout the titration. The vacuum-tube voltmeter, like electron-tube titrimeters, requires a power line source of alternating current of a very good degree of regulation. Moreover, the apparatus needed for both methods is costly. Consequently, an electrometric method of titration that will overcome these disadvantages is desirable. An inexpensive apparatus was developed which mas found to be applicable to electrometric titrimetry to the same extent as the more expensive equipment. The object of this paper is to explain the operation and applications of this titrimeter, especially its application to simplifying the Schollenberger method of determining organic matter, as employed in the laboratory of Project 4 of the American Petroleum Institute. The instrument gives promise of being useful in other types of titrations, and three others are described in detail in this paper-namely, ferrous
