Microdetermination of Sulfate
A
Colorimetric Estimation of the Benzidine Sulfate Precipitate
B E R N A R D KLEIN, First Lieutenant, Snitary Corps, Laboratory Service, Tilton General Hospital, Fort Dix, N. J.
I
S COKNECTION with another investigation, the need arose
for a simple, rapid, and accurate method for the determination of small amounts of sulfate, using only the limited equipment and reagents t’hen available. The following method was developed and proved extremely satisfactory: The sulfate in the sample was precipitated as the benzidine sulfate, purified, and dissolved in 0.2 N hydrochloric acid, diazotized, and coupled, after destroying the excess nitrous acid, with Bratton and hlarshall’s reagent (I), N-( 1-naphthy1)ethylencdiamine dihydrochloride. The resultant intense purple color was read in a KlettSummerson photoelectric colorimeter. Colorimetric methods for the determination of sulfate are not new. Yoshimatsu (8) first treated the dissolved benzidine sulfate precipitate with a mixture of iodine, potassium iodide, and ammonia, and compared the resultant brown color Kith a benzidine sulfate standard similarly treated. Hubbard ( 2 ) and later Wakefield ( 7 ) used the yellow color produced by the action of hydrogen peroxide and ferric chloride on the dissolved benzidine sulfate precipitate. Kahn and Lieboff (3),thus far, were the only ones to couple diazotized benzidine sulfate with an alkaline solution of phenol. Lentonoff and Reinhold (4) used the color produced by the action of sodium ~-naphthoquinone-4sulfonate on an alkaline solution of benzidine sulfate as a basis for comparison. Since its initial introduction by Bratton and Marshall ( 1 ) as a coupling agent for the determination of sulfonamides, N-(1naphthyl)et,hylenediamine dihydrochloride has found increasing popularity. Shinn (6) found that it gave superior results for the determination of nitrite in water, sewage, and foods. Recently, Looney and Dyer have used this reagent with success in the determinat,ion of potassium in serum (6). REAGENTS
The walls of the tube are washed with 2.0 ml. of acetone-alcohol in a manner to avoid disturbing the tightly packed precipitate. The tube is recentrifuged for 5 minutes, and the su ernatant poured off and permitted to drain for 5 minutes on the fiyter paper pad. The washing and draining procedure is repeated once more. Two milliliters of 0.2 N hydrochloric acid are blown into the centrifuge tube. The precipitate dissolves almost instantly. The tube is placed in an ice-water bath, 1.0 ml. of sodium nitrite solution is added, and the tube is shaken to mix the solutions and allowed to stand for 3 minutes. One milliliter of ammonium sulfamate solution is added, and the tube is shaken and allowed to stand for an additional 2 minutes. Finally, 1.0 ml. of N-(lnaphthy1)ethylenediamine dihydrochloride solution is added to develop the color. After mixing, the violet solution is permitted to stand for 20 minutes, transferred quantitatively to a 50-ml. volumetric flask, and diluted to mark with distilled water. The solution is read in a photoelectric colorimeter using a green filter. The Klett S o . 54 is satisfactory for the Klett-Summerson instrument. It is recommended that a standard solution of benzidine hydrochloride or standard sulfate solution containing 0.03 mg. as sulfate be carried through, a t the same time. The former solution need only be diazotized and coupled, while the latter must be carried through the entire procedure as described above. A blank, using 2.0 ml. of 0.2 N hydrochloric acid, diazotized and coupled witfi the color reagent, serves as zero reading on the photometer. Table I.
so4
Error
%
MQ.
0.0150 0.030 0.030
0.0152 0.0300 0.0303
0.0002
1.3
0.060
0.0003
0.060
0 1.0 0
0.060
0.0597 0.0921 0.1470
0.0003 0.0021 0.0030
0.5 2.3 2.0
0.150
MQ.
Error
MQ.
0.090
Sodium nitrite, O.l%, prepared fresh daily. Ammonium sulfamate, 0.5%. N-(1-naphthy1)ethylenedianline dihydrochloride, E a s t m a n Kodak Company No. 4835, 0.lg The solution is made up in distilled water and kept in a dark%ottle. The reagent keeps best if refrigerated. Benzidine hydrochloride reagent, 4.0 grams of pure benzidine hydrochloride dissolved in a small amount of distilled n-ater and matlc up to 250 ml. with 0.2 N hydrochloric acid. Standard Sulfate, Solutions. Stock, 0.5437 gram of reagent potassium sulfate dissolved in distilled water and diluted to 1 liter; 1.0 ml. is equivalent to 0.3 mg. of sulfate. Dilute standard, 10.0 ml. of the stock standard diluted to 100 nil. with distilled water; 1.0 ml. is equivalent to 0.03 mg. of sulfate. Standard Benzidine Hydrochloride. Stork, 0.4014 gram of pure benzidine hydrochloride dissolved in a small amount of 0.2 N hydrochloric acid and diluted to 100 ml. Kith the same solvent; 1.0 ml. is equivalent to 1.5 mg. of sulfate. Dilute standard, 1.0 ml. of stock solution diluted to 100 ml. with 0.2 it’ hydrochloric acid; 1.0 ml. is equivalent to 0.015 mg. of sulfate. Acetone-ethanol, 1 to 1, using 9570 ethanol and reagent acetone.
Determination of Sulfate
SO, Recovered
CALCULATION
Reading of unknown + reading of standard X concentration of standard = mg. of sulfate in sample DISCUSSION
This method offers many advantages. The ease and simplicity u.ith which the determination is carried out, together with the availability of the reagents used, are foremost. K O extreme precautions are ncccssary beyond attontion t o the use of narrowed conical tips on the centrifuge tubes. These are readily fashioned by draiTing the tips of ordinary glass centrifuge tubes in a name until the diameter of the tip is about 2 mm. After centrifugation, thc precipitated benzidine sulfate packs tightly and can be casily washed without disturbance or loss. Tubes which have been soaked in dichromate-sulfuric acid mixture must be carefully washed to remove all traces of sulfate. The color obtained on final coupling with Bratton and hlarshall’s reagent has remarkable stability. I n initial runs, stability studies revealed no decrease in intensity even after 12 hours. The color fol1ou.s Bcer’s law closely. This is true of both solutions of pure benzidine hydrochloride and known sulfate solutions used in recovery studics. The precision of triplicate detcrminations was excellent, not varying by more than 0.1%. The limitations of benzidine sulfate precipitation in the presencc of large amounts of chloride and phosphate should he pointed out. At the time this study was carried out the sulfate conccntration in a dialyzate which had no, or at most, traces of chloridc
PROCEDURE
T o a 15-ml. centrifuge tube with a narrowed conical bottom, containing 1.0 ml. of glacial acetic acid and 1.0 nil. of benzidine hydrochloride reagent, an amount of sample containing between 0.015 and 0.15 mg. of sulfate is added, the solution is mixed, 2.0 ml. of acetone-alcohol are added, the tube is capped, and the contenls are thoroughly mixed by rolling between the hands. The tube is allowed to stand in an ice-water bath for a half hour to complete precipitation. At the end of this period, the tube is centrifuged a t 2500 r.p.ni. for 10 minutes and the supernatant poured off with one rapid motion. While still inverted, the mouth of the tube is carefully touched with a pad of clean filter paper to blot off excess liquid. The tube is permitted to drain on the filter paper pad for 5 minutes.
536
August, 1944
ANALYTICAL E D I T I O N
and phosphate present was being checked. This provided no interference. I n the case of biological materials, phosphate should be completely removed. Chloride should not be present in amounts such that the weight ratio of chloride to sulfur exceeds 30; else, precipitation of benzidine sulfate is not complete. The method is accurate in a range extending from 0.05 to 0.150 mg. of sulfate with a maximum error of 2%. The results obtained on pure sulfate solutions are summarized in Table I. Each value is the mean of triplicate determinations.
537 LITERATURE CITED
(1) Bratton, A. C., and Marghall; E. K., J . Biol. Chem., 128, 537 (1939). (2) R *s.,Ibid**74, (lgZ7). (3) Kahn, B.S.,and Lieboff, S.L., Ibid., 80, 623 (1928). J. G,,Ibid., 114, 147(1936). (4) Lentonoff,T,V,,and (5) Looney, J. M., and Dyer, C. G., J . Lab. Clip. Md., 28, 355 (1942). (6) Shinn, M., IND. ENQ.cHEM,, A N A L . ED.,13,33 (1941). (7) Wakefield, E.G., J . Bi02. Chem., 81,713 (1929). (8) Yoshimatsu, 5 . I., Tdhoku J . Ezpt. Med., 7,553 (1926). Hubbardl
NOTES ON ANALYTICAL PROCEDURES Improved Distillation Receiver
A n Observation of Possible V a l u e
HOWARD M. WADDLE1
for Sugar Determinations
State Engineering Experiment Station, Georgia School of Technology, Atlanta, G a .
DANIEL LUZON MORRIS, The Putney School, Putney, V t .
I
S AT\’ attempt to modify the Sichert and Bleyer reagent (1)
for micro use, potassium iodide was included in the reagent. When this reagent was heated with glucose, a precipitate formed which was identified as cuprous iodide. The presence of the iodide ion speeds up the reduction reaction, presumably because of the removal of the cuprous ion from solution as cuprous iodide. The observation may have value for the determination of sugars, for the cuprous iodide can be determined iodometrically in the solution or separated and weighed directly. I t s weight is of the order of ten times that of the glucose taken. The reagent may be made up as follows: To 250 cc. of water are added 500 grams of hydrated sodium acetate, 75 cc. of 5% acetic acid, and 5 grams of potassium iodide. The solids are dissolved by warming the solution, and 25 grams of crystalline cupric sulfate are added as a 10% solution. About 40 grams of glucose are now added, the volume is made up to 1 liter, and the mixture is heated in boiling water for 45 minutes, let cool slowly, and filtered to remove the crystalline precipitate of cuprous iodide which had separated during the heating. For the determination, the solution is heated for 30 minutes a t 100” with an equal volume of glwose solution. Further work on this reagent is not contemplated. Lowering of the pH by the addition of more acetic acid results in the formation of less precipitate for a given amount of glucose; if the amount of acetic acid is decreased, a white precipitate (apparently cupric hydroxide or a basic cupric salt, as it is nonreducing) is formed on heating, even in the absence of glucose. Omission of the preliminary heating with glucose causes a slight black precipitate to form during the determination if the quantity of glucose being determined is very small. The use of more than 5 grams of potassium iodide per liter increases the speed of the reaction, but makes iodometric estimation of cuprous iodide in the solution difficult. Less than 0.2 gram of glucose will give no reduction. The reagent is affected by maltose, whose reduction, under the conditions mentioned, is about l / g that of an equal weight of glucose. The maltose reduction is not complete even after 50 minutes of heating, whereas the glucose reductionshows no increase after 30 minutes.
THE
uiual variety of cow’s-udder fraction cutter ( 1 ) has the inherent disachantage that each drop of distillate must be collected in one of the receivers attached to the cutter. The modification described makes it possible to interrupt the flow of a distillate while the operator follows the course of a temperature change in the fractionating column. Since the fraction cutter can be clamped in a stationary position, it is possible, in the caie of low-boiling fractions, to surround the entire receiver with a freezing mixture. FROM DISTILLING FLASK
In practice bulb F is approximately 10 mi. in diameter. Ground joint C has a standard taper 14/35, and the body, A , is made from a 50-ml. Erlenmeyer flask. All glass tubing, B , is 8 mm. The inside rod that conducts the distillate to the receivers must rotate freely and must not leave the surface of the tube coming from stopcock D, 2-mm. bore. LITERATURE CITED
LITERATURE CITED
(1) Sichert and Bleyer, 2.anal. Chem., 107,328 (1936). Txie work waa aided by a grant iron] hIead Johnson and Co.
(1) Reilly and Rae, “Physico-chemical Methods”, 3rd ed., Vol. I1 p. 94, New York. D.Van Nostrand Co., 1939. 1 Preaent address, Rwearch Divieion, West Point Manufacturing Co., Shawmut, Ala.
