662
ANALYTICAL CHEMISTRY
permit work with as small sample sizes as could be weighed with available balances or, on the other hand, the determination with normal sample sizes of sulfur as an impurity or as a minor constituent of the large molecules of biological importance. Undoubtedly the greatest interest in the technique will evolve from the obvious possibilities for streamlining it into a rapid, titrimetric procedure. The following three modifications may be suggested for those interested in this development. The time-consuming Carius digestion could be replaced with the rapid catalytic combustion procedure using alkaline peroxide as absorber. Lead sulfate would be brought down in the acidified absorber, separated by suction filtration (filterstick immersed in the large Pregl test tube), and washed and dissolved in acetate buffer-all in the same vessel. A rotating platinum microelectrode could replace the dropping mercury electrode giving better stirring during the titration and facilitating the ‘ attainment of equilibrium readings after each addition of titrant. The practice of recording the titrant volume a t the point of minimum galvanometer reading as the end point is not a justifiable expedient. This practice can never approach in either precision or accuracy the results by the more tedious graphical line intersection technique. The handling of the data in the latter case, however, can be expedited somewhat by determining a correction factor graphically or from a table.
Table I. Results by Amperometric-Carius Sulfur Method % Sulfur Compound Cysteine hydrochloride
Theoretical 20.33
Sulfanilic acid
18.51
.Illy1 thiourea
27.60
Found 20.38 20.14 20,22 18.58 18.63 18.47 27.51 27.38 42 70 11.38
Thiourea 42.615 Benzidine sulfate 11.35 Cystine 26.69 26.77 a Theoretical for thiourea is 42.12%. The figure shown is the result of a careful gravimetric assay using 20-mg. samples.
LITERATURE CITED ( 1 ) Kolthoff, I. M.,and Pan, (1939). (2) Ihid., 62, 3332 (1940).
T.D., J . Am. Chem.
Soc., 61, 3402
(3) Rulfs, C . L.,-4x.1~. CHEM.,19, 1046 (1947). RECEIVED for review July 21, 1952. Accepted December 3, 1952
Modification of the Determination of Urea by the Diacetyl Monoxime Method HOWARD S. FRIEDMAN, T a m p a Municipal Hospital, Tampa, Fla. of the quantitative Fearon reaction for urea in blood and other body fluids is presented. The modified procedure eliminates the objectionable odors of fuming strong hydrochloric acid. Using a 1 to 20 filtrate of blood, or a 1to 200 dilution of proteinfree urine, and a Coleman Junior spectrophotometer, the method is applicable to blood concentrations between 5 and 500 mg. of urea nitrogen per 100 ml. of whole blood, and to urine concentrations 10 times those given for whole blood. Fearon ( 1 ) observed that solutions of urea and LV-substituted ureas yielded colored solutions when heated with diacetyl monoxime in strong acid solution, following color development with potassium persulfate. He found that only unsubstituted urea gave a yellow color in this procedure. Ormsby ( 4 ) has proposed a quantitative procedure for urea in blood and other body fluids, using a tungstic acid filtrate, heating with diacetyl monoxime, and strong hydrochloric acid and developing the color with potassium persulfate. Satelson and his associates (5)have shown that the potassium persulfate serves to destroy the hydroxylamine formed during heating. I n the absence of persulfate, hydroxylamine inhibits color formation. Xatelson proposed the use of free diacetyl, since this appeared to be the active reagent. I n the absence of hydroxylamine, persulfate had no effect on the color development. Kawerau ( 2 ) suggested the use of arsenichydrochloric acid mixture for simultaneous color development with diacetyl monoxime in dilute acetic acid solution, the hydroxylamine being oxidized as it is liberated during the heating period. Attempts in this laboratory to reproduce a standard curve by the method of Natelson have met with no success. The reaction is apparently quite photosensitive, as mentioned elsewhere ( 3 ) . Furthermore, the reaction gives rise to the particularly objectionable odor of free diacetyl during the heating period. An attempt to adapt the method of Kawerau met with a similar objectionin this case, the odor of strong fuming hydrochloric acid during the heating period. The author decided to try a solution of arsenic acid in 1 to 1 sulfuric acid, which is approximately of the same normality as concentrated hydrochloric acid.
REAGENTS AND METHOD
RIODIFICATIOX
l
!
A Somogyi filtrate ( 5 )was used, permitting the determination of both urea and glucose on the same filtrate, a procedure being aidely adopted a t the present time. The final dilution was 1 to 20, instead of the usual 1 to 10. The reagents used were those of Kawerau, (%’) with the esception of the arsenic acid solution, which was prepared in 1 to 1 sulfuric acid instead of concentrated hydrochloric acid. The spectrophotometer used T+ as the Coleman Junior, Model 6A. The light path was 19 mm., using 19 X 150 mm. cuvettes. The wave length selected was 475 mp, as indicated by Kawerau ( 8 ) . Although Satelson ( 3 )suggestFd the use of 480 mp, there appears to be no practical significance in this instance,.since the Coleman instrument is incapable of a spectral band narrower than 35 mp. Reagents. ZINCSULFATE,5% Dissolve 50 grams of zinc sulfate heptahydrate (Baker’s analyzed grade) in about 700 ml. of distilled water in a 1000-ml. volumetric flask. After solution is complete, dilute to the mark with distilled water, mix well by inversion, and store in a borosilicate glass reservoir bottle, connected to a refillable 50-ml. buret. BARIUM HYDROXIDE, DILUTED. Prepare about 1000 ml. of a saturated solution of barium hydroxide octahydrate (Baker’s analyzed grade), using freshly boiled distilled water. Filter while hot into a bottle of capacity slightly greater than 1000 ml., preferably \+ith a screw cap. When the solution has cooled to room
Table I. Comparison of Modified Diacetyl Monoxime and Karr Urease Procedures - 31g. of Urea Nitrogen per 100 hll. of Blood Diacetyl monoxime method 10.6
13.3 20.3 18.0 15.5 11.7 12.3 15.5 14.0 14.0 4v. 14.5 St. dev. + 2 . 8
Karr urease method 10.0 14.0 20.0 17.0 16.0 12.0 12.5 15.0 14.5 13.5 .4v. 14.5 St. dev. +2.6
Difference +0.5
-0.7 +0.3 $1.0 -0.5 -0.3 -0.2
Av.
t0.5 -0.5 +0.5 0.5
St. dev. rtO.2
%
Deviation +5
-5 +1.5 +5.9 -3.1 -2.5 -1.6 +3.3 -3.5 +3.7
663
V O L U M E 25, N O . 4, A P R I L 1 9 5 3 temperature, and any additional barium carbonate has settled to the bottom, titrate 4 ml. of the 5y0zinc sulfate solution with the concentrated barium hydroxide solution, using phenolphthalein as the indicator. Dilute the barium solution so that between 13.5 and 14.5 ml. are required to titrate 4 ml. of 5% zinc sulfate solution. Store the dilute barium hydroxide in a large reservoir bottle connected to a 100-ml. refillable buret. Attach carbon dioxide absorption bulbs to both the open end of the buret and to the air inlet of the reservoir bottle. ;IRSENIC-SULFURIC ACID. With careful coolingandmixing,pour 500 ml. of concentrated sulfuric acid (Baker’s analyzed grade) into 500 ml. of distilled water in a 2000-ml. Erlenmeyer flask. After the solution has cooled to about 50” C:, add 100 grams of arsenic acid (Baker & Sdamson, Reagent), mix well, and allow to cool. Store in a chemically resistant glass-stoppered bottle. This is enough reagent for approximately 1000 tests. DIACETYL ;\IOXOXIME (EASTMAN), 1% in 5y0 acetic acid (Baker’s Analyzed grade). Five hundred milliliters of this reagent is enough for approximatply 1000 tests. Table 11.
Recovery of Urea Nitrogen from Blood
hfg. of Urea h’itrogen per 100 M1. of Blood Added Found Recovered Difference ... 10.3 2:0 0.0 12.3 4.2 +0.2 14.5 4 16.3 6 6.0 0.0 +0.2 8.2 18.5 8 i.n. 10.4 f0.4 20.7 11.7 -0.3 12 22.0 14.0 14 0.0 24.3 +0.2 16.2 26.5 16 -0.3 17.7 28.0 18 20.0 0.0 30.3 29 AT. 0 . 2 1 0 . 1
4
% Deviation
... 0
+5
0 +3 +4 -2.5 0 +1.2 -1.7 0
Preparation of Filtrate. Using an Ostwald-Folin pipet, place 1 nil. of whole blood in a small 50-ml. narrow mouth container. (Two-ounce glass jars are used in this laboratory for the preparation of all filtrates.) Add from a buret 10 ml. of distilled watpr. RIix by gentle swirling. Add 7 ml. of dilute barium hydroxidp solution from the buret, swirl, and let stand about 30 seconds. ( A convenient method of timing is to add the dilute barium solution to about six samples. X h e n the sixth addition is completed, the first sample will be ready). Add 2 ml. of zinc sulfate solution to each sample, Close each bottle M ith a rubber stopper, and shake vigorously until the mixture is smooth and fluid. There should be no bubbles present. Filter into a second 50-ml. container, using a 9-cm. Khatman #1 filter paper. Procedure. Place 1 ml. of the 1 to 20 filtrate in the bottom of a tall trst tube (15 X 200 mm.), graduated a t 10 and 20 ml. Add 0.5 nil. of the diacetyl monoxime reagent, and mix by lateral shaking. Just before placing in a boiling-water bath, add 1 ml. of arsenic-sulfuric acid mixture, and mix by lateral shaking. Place in a boiling-water bath for exactly 10 minutes. Remove and let cool a t room temperature for esactly 3 minutes. Dilute to 10 nil. 1%ith distilled water, mix by inversion, and transfer to Coleman cuvettes. Read within 7 minutes after the addition of the water. Prepare a blank by substituting 1 ml. of distilled water for the filtrate. Set a t 0 absorbance. (For urine, in the absence of protein, use 1 ml. of a 1 to 200 dilution with distilled water, multiply the final result by 10). The standard curve was established from a standard urea solution containing 2.14 grams of urea, or 1.00 gram of urea nitrogen per liter. This stock solution was diluted with distilled water so as to obtain solutions equivalent to 1 to 20 dilutions of filtrates containing 0, 10, 20, 30, 40, and 50 mg. of urea nitrogen per 100 ml. The readings obtained from the standard solutions are shown in Figure 1. DISCUSSION
It can be seen from Figure 1, that the curve is a straight line from 10 mg. per 100 ml. equivalent, or slightly below, up to 50 mg. per 100 ml. equivalent. Below 10 mg. and above 50 mg. as determined with additional solutions equivalent to 1, 2, 3, 4, 5, 6, 7, 8, 9, 60, and 70 mg. per 100 ml., the curve tends to flatten. As set up in this laboratory, 1 mg. of urea nitrogen per 100 ml. equivalent gave an absorbance of 0.013 in the flat portion of the curve, which is easily readable. Since there is little clinical significance in an accuracy better than 1 mg. of blood urea nitrogen per 100 ml. the method is sufficiently accurate for these
purposes. Between 5 and 10 nig. per 100 ml. equivalent, 1 nig. of urea nitrogen gave an absorbance of about 0.010. This extends the usefulness of the curve down to 5 mg. per 100 ml. equivalent. The range can be extended beyond 100 mg. if the following is kept in mind. hfter the analyst has had some experience with the method, it is possible to recognize those samples which after dilution to 10 ml. will give an absorbance greater than that for 50 mg. as determined in the particular laboratory. The sample must be diluted to 20 ml., and the final result multiplied by two. If this solution still reads above the upper limits of the curve, 2 1111. of the solution are taken from the cuvette and placed in a similar cuvette. Eight milliliters of distilled water areadded; thesolution is mixed, and results are read in the spectrophotometer. The result obtained must be multiplied by two. These manipulations must be done within the 7-minute period after the initial dilution. I n 0.5 ml. of 1% diacetyl monoxime solution there are 5 nig. of diacetyl monoxime, and in 1 ml. of a 1 to 20 filtrate of blood containing 1000 mg. of urea nitrogen per 100 ml., there are 1.07 mg. of urea. Since it is improbable that more than two molecules of diacetyl combine with one molecule of urea, there is sufficient diacetyl present for the determination of 100 mg. of urea, or 470 mg. of urea nitrogen per 100 ml. If the diacetyl monoxime solution is kept in the refrigerator, it is stable for 4 to 6 months.
k Oe70
1
0.50
YZ
P
‘
0.30
0
0
10 PO 30 40 50 60 MG. OF UREA N PER 100 ML. OF WHOLE BLOOD
Figure 1.
Readings from Standard Solutions
A comparison of the modified procedure with the Karr urease method, using Nessler’s solution, is shown in Table I. Values given are the average of three determinations, and represent urea nitrogen. Recovery experiments were run using standard amounts of urea solution added to portions of a pooled specimen of whole blood. The results are shown in Table 11. CONCLUSIONS
.
Above an absolute concentration of 2.5 micrograms of urea nitrogen per ml. of final solution, the curve levels off sharply. This agrees with most of the reports on this reaction. However, by proper dilution of solutions containing greater concentrations, a value can be obtained for absorbance which falls within the range covered by Beer’s law. Further, since the blank solution has an absorbance of less than 0.010, it is not necessary to dilute
ANALYTICAL CHEMISTRY
664
it when an unknown is diluted. Since solutions to be diluted contain more than 50 mg. per 100 ml. equivalent, and have an absorbance greater than 0 . 5 i 5 , the error from this source is less than 27,. Below 0.5 microgram per ml. of final solution, corresponding to 10 mg. per 100 ml. of whole blood, the applicability of Beer’s law diminishes rapidly, until below 0.25 microgram per ml. the curve is quite unreliable, probably owing to the magnification of small errors. The upper limit of usefulness is determined by the amount of diacetyl monoxime present in the test solution. It is probable t h a t the range can be extended to a t least 500 mg. of urea nitrogen per 100 ml. of whole blood by proper dilution of the final solution.
If a calibration curve is set up from standard urea solutions, covering the range of 5 to 50 mg. of nitrogen per 100 ml., by steps of I mg. per 100 ml., the differences in absorbances of adjacent readings will be 0.010 or slightly more. This is more than sufficient to give a sensitivity of 1 mg. of nitrogen per 100 ml. A greater sensitivity would not be of clinical value LITERATURE CITED
(1) F e a r o n , 17.R., Biochem. J . , 33, 902 (1939). (2) K a w e r a u , E., Sci. Proc. Roy. Dublin Soc., 42, 63 (1946). (3) S a t e l s o n , S.,S c o t t , hI. L., and Beffe, C., .4m. J . Clin. Pathol., 21, 275 (1951). (4)Ormsby, A . A,, J . B i d . Chem., 146, 596 (1942). (5) Somogyi, XI.,J . Biol. Chem., 86, 655 (1930).
RECEIVED for review September 25%1952. Arcepted December 3, 1952.
The Determination of Copper in Mineral Oils Using an Ion-Exchange Technique HERBERT BUCHWALD
OPPER
AND
L. G. WOOD. Manchester Oil Refinery, Ltd., Manchester, England
compounds can be quantitatively removed from min-
C eral oils by adsorption on a cation-exchange resin and sub-
sequently recovered by acid washing. The copper in the acid solution, free from organic contamination, can then be determined by any of the standard methods. In the present instance copper contents as low as 0.1 p.p.m. have been determined colorimetrically. In an investigation requiring the determination of the copper content of a large number of samples of used transformer oil it \vas found that the generally accepted methods were excessively lengthy, and were not capable of application to the low concentrations of copper encountered with any degree of repeatability. T h e standard methods ( I , 5 ) involve either lwt or dry ashing of the sample, followed by the colorimetric determination of the copper content of the ash. In the former method the procedure was lengthy, requiring the oxidation of relatively large quantities of organic matter, and in the latter, loss of copper compounds by volatilization lvas a distinct possibility.
concentration of copper on a cation-exchange resin, and subsequent polarographic analysis of the solution. Frizell (4) and Wickbold (13, 14) have discussed the use of ion-exchange resins for analytical purposes, while Swanson (22) has mentioned unpublished work carried out a t Shell Thornton Research Centre on the recovery and determination of iron and copper from used engine oils by an ion-exchange technique. It was found that if a cation-exchange resin containing sulfonate groups was converted to the hydrogen form and then washed with 2-propanol to remove water, it would remove dissolved copper compounds from mineral oil quantitatively. Oil could then be washed from the resin by percolation of a further quantity of 2-propanol, which could in turn be removed by percolation of distilled xater. The copper could be recovered quantitatively by percolation of dilute sulfuric acid down the column. The copper content of the acid percolate could then be determined colorimetrically. APPARATUS
Table I.
Copper Content of Solutions of Copper Yaphthenate i n Mineral Oil
Copper, P.P.M. Added Found 57.50 56.90 11.80 11.30 11.80 11.30 5.90 5.40 1.65 1.10 1.60 1.10
Blank, P.P.hI. 0.55 0.55 0.55 0.55 0.55 0.55
Copper Found less Blank, P.P.M. 56,95 11.25 1?,!25 a . 35 1.10 LO5
Massey (8) has shown that copper compounds can be quantitatively removed from mineral oil by sulfuric acid washing, and the concentration of copper can be determined colorimetrically by the sodium diethyldithiocarbamate method a t concentrations as low BS, 0.05 p.p.m. Kreulen (7) used an acid washing technique involving refluxing the sample with 10% sulfuric acid followed by removal of the acid layer. Both methods suffer from the disadvantage that a small amount of organic matter is always present i n the acid layer after separation, and has to be removed by vigorous oxidation or wet ashing. Various authors have proposed methods depending upon w-et ashing or acid extraction ( I O ) , followed by titration with dithizone ( 2 ) or amperometric titration (9). Cranston and Thompson (3) have shown that traces of copper in milk products can be determined by removal of organic matter,
A chromatographic column approximately 35 cm. long and 1 cm. in diameter with an upper reservoir of 100 ml. capacity was used. The lower end of the column was closed by a stopcock, and the resin was held in place by a plug of glass wool. .4 “Spekker” photoelectric absorptiometer was used for the colorimetric determination of the recovered copper. REAGENTS
Sulfuric acid, 10% v/v, il.R. grade Ammonium hydroxide, 20% v/v, A.R. grade iimmonium hydroxide, “880”, A.R. grade Ammonium citrate solution. Five-hundred milliliters of A.R. grade “880” ammonium hydroxide and 500 grams of -4.R. grade citric acid were diluted to 1 liter with distilled water. Gum acacia solution, 0.5%, in distilled water, filtered before use Sodium diethyldithiocarbamate solution, 0.2% in distilled water, freshly prepared 2-Propanol, absolute, redistilled Indicator paper, B.D.H. “Wide range” Ion-exchange resin, “Zeo-Karb” 215 PROCEDURE
The column was filled with a slurry of the resin in distilled water to give a bed approximately 30 cm. deep. The level of liquid was never permitted, a t any time, to fall below that of the resin. Percolation of 50 ml. of 10% sulfuric acid through the column over a period of 15 minutes ensured that the resin was in the hydrogen form. Before the acid level had fallen below that of the resin, the column was washed with successive 10-ml. ortions of distilled water until the effluent had a pH of about 4 ($etermined
