Rapid Turbidimetric Method for Determination of Sulfates I
JOSEPH F. TREON AND W. E. CRUTCHFIELD, JR. Kettering Laboratory of Applied Physiology, College of Medicine, University of Cincinnati, Cincinnati, Ohio
No. 12 paper, and filtered by suction. As soon as the filtration is complete, the paper is washed three times with 15 to 20 ml. of distilled water. Three drops of alcoholic methyl red are added to the filtrate, which is made faintly alkaline with approximately N sodium hydroxide and then the acidity is regulated by making it just acid with hydrochloric acid (1part of concentrated hydrochloric acid plus 4 parts of water). An excess of 1 ml. of hydro4) is added and the solution is diluted to 250 chloric acid (1 ml. with distilled water in a volumetric flask and shaken. In order to eliminate any slight variation caused by the color of the urine, just enough solution is withdrawn for a blank (about 1.5 ml.) and read in a 1.25-cm. (0.5-inch) precision cell on the neutral wedge photometer, with a 540-millimicron filter (Aminco No. 54). To the remaining solution in the 250-ml. volumetric flask is added 1 gram of 20- to 30-mesh barium chloride. (This may be prepared from reagent-grade barium chloride, or obtained already screened from the Parr Instrument Company, Moline, Ill.) As a matter of convenience, a scoop may be made which will deliver 1 gram. The solution is shaken well, placed in the same cell, and read immediately on the neutral wedge photometer at 540 millimicrons. This reading is converted to the amount (in milligrams) of sulfate in 7.5 ml. of urine by reading from the standard curve (Figure 1). HYDROLYSIS OF ETHEREAL SULFATES AND DETERMINATION OF TOTAL INORGANIC AND ETHEREAL SULFATE.A 7.5-ml. sample of urine is pipetted into a 150-ml. Erlenmeyer flask containing 2 ml. of water and 7.5 ml. of hydrochloric acid (1 4),and a coldfinger condenser is inserted into the top of the flask. The solution is boiled for 20 to 30 minutes, then cooled. Three drops of alcoholic methyl red are added to the sample. It is made just alkaline with approximately N sodium hydroxide and is filtered by suction through a Whatman KO.12 paper: in a small Buchner funnel. The paper is washed three times with 15 to 20 ml. of distilled xater. The filtrate is made just acid with hydrochloric acid (1 4). An excess of 1 ml. of hydrochloric acid (1 + 4) is added, and the solution is diluted to 250 ml. with distilled water
S
IKCE benzene, phenols, and various other cyclic com-
pounds alter the ratio of inorganic t o ethereal (organic) sulfates in urine, it seemed desirable to obtain a rapid method for the determination of this ratio. Gravimetric methods have been used largely for this purpose but they are timeconsuming. A nephelometric method for determination of inorganic sulfates in biological fluids has also been reported recently by Medes and Stavers ( 2 ) .
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FIGURE1. DIFFERENCES BETWEEN WEDGE READINGS FOR SAMPLES WITH VARYING CONCENTRATIONS OF SULFATE AND BLANKS. 0.5-INCH C E L L U S E D
The authors first devised a colorimetric method ( I ) ,based on precipitation of the sulfate with benzidine and coupling of the benzidine with chromotropic acid to yield Dianil Blue R (colour index No. 390, 3). Since i t proved to be somewhat tedious, however, the simpler and shorter photometric method here reported was developed. The advantages in this method over gravimetric methods are: (1) the speed of determination is increased; (2) a small aliquot may be used; (3) for the final measurement of the sulfates there is a choice of several instruments-namely, the neutral wedge photometer, the spectrophotometer, and the Leitz clinical photoelectric colorimeter.
Procedure
CONGENTR4TIOH (HO SULFITE IN 7.5 ML URINE]
FIGURE 2. SPECTROPHOTOMETRIC DENSITY READINGSWITH
DETERMINATION OF INORGANIC SULFATES I N URINE. The urine is collected, the total volume is measured, and 7.5 ml. are pipetted into a small Bdchner funnel equipped with Whatman
VARYING
CONCENTRATIONS
OF
SULFATE.
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0.6-INCH
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in a volumetric flask and shaken after which the procedure for the determination of inorganic sdfates is followed. PREPARATION OF STANDARD CURVE. The standard curve is PrePared from a series of readings on known amounts of sulfate in ‘synthetic urine” solutions prepared as described by Cholak (I), and containing approximately 5 mg. of sulfate per milliliter. The exact amount of sulfate is determined either by direct weighing of anhydrous sodium sulfate (Merck reagent) or by titratin diluted sulfuric acid (sulfate equivalent to acid) against standarf alkali. To 7.5 ml. of synthetic urine is added 1 ml. of the standard sulfate solution described above. In a similar manner solutions are made with 0.0 2.0, 3.0, 4.0, 5.0, and 6.0 ml. of standard sulfate. They are made just alkaline to alcoholic methyl red with a p proximately N sodium hydroxide, and then just acid with hydrochloric acid (1 4),after which an excess of 1ml. of hydrochloric acid (1 4) is added. The solutions are washed thoroughly into 250-ml. volumetric flasks, diluted to the mark, and shaken. The procedure is continued as described for determination of inorganic sulfates in urine. The differences between the wedge readings for samples and blanks are plotted against the concentratlon (milli4rams of sulfate in 7.5 ml. of urine) to give a standard curve (Figure 1).
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CALCULATION OF PER CENT OF INORGANIC SULFATES IN URINE. The amount of inorganic sulfates in the entire sample is calculated as follows: total volume of urine Milligrams of sulfate as read from curve X 7.5 quantity of inorganic sulfates (in milligrams)
~ P d o 9 0 s b 7 0 d 5 0 4 L d IO
FIGURE 3. READINGSWITH VARYINGCONCENTRATIONS OF SULFATE, OBTAINEDWITH LEITZ COLORIMETER. STANDARD LEITZCELLUSED
Inorganic plus ethereal sulfates in the entire sample: total volume of urine Milligrams of sulfate as read from curve X 7.5 quantity of inorganic plus ethereal sulfates (in milligrams) Per cent of inorganic sulfates in urine: Milligrams of inorganic sulfate as read from curve x 100% = Milligrams of inorganic ethereal sulfate as read from curve % inorganic sulfates in urine
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TABLEI. CONPARISOX OF SPECTROPHOTOMETRIC AND GRAVIMETRIC METHODS (Findings in 100 ml. of urine) Inorganic Inorganic
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Source of Urine
Method Used
Normal mixed human Turbidimetric Gravimetric Rabbit 243 Turbidimetric Gravimetric Rabbit 244 Turbidimetric Gravimetric Rabbit 245 Turbidimetric Gravimetric Rabbit 246 Turbidimetric Gravimetric Rabbit 241 Turbidimetric Gravimetric Rabbit 242 Turbidimetric Gravimetric Rabbit 248 Turbjdimetric Gravimetric Rabbit 249 Turbidimetric Gravimetric Rabbit 245 Turbidimetric Gravimetric Rabbit 246 Turbidimetric Gravimetric Rabbit 268 Turbidimetric Gravimetric Rabbit 269 Turbidimetric Gravimetric Rabbit 270 Turbidimetric Gravimetric Rabbit 271 Turbidimetric Gravimetric
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d
LElTZ COLORIMETER REAMNGS
Ethereal Inorganic Ethereal Inorganic Inorganic BaOS, Bas04 so4-- so,-604-Mg. Mg. MQ. MQ. % 148.7 139.3 93.6 356.8 327.2 147.8 134.7 91.1 98.0 89.3 91.1 227.6 202.6 93.7 83.4 91.5 156.0 134.6 86.3 385.0 332.0 158.4 136.3 86.0 106.6 95.3 89.3 240.0 212.0 98.8 87.0 88.0 157.3 153.3 97.4 416.0 363.0 171.2 149.4 87.0 192.0 167.3 81.9 445.0 343.5 183.1 141.1 77.2 78.0 73.3 94 0 155.2 141.2 63.9 57.7 92.0 174.0 157.3 90.4 430.4 378.0 177.1 155.6 87.9 189.3 170.6 90.1 429.2 400.4 176.6 164.7 93.3 157.3 134.6 85.6 406.5 350.5 167.3 144.0 86.0 133.3 108.0 81.0 309.0 273.0 127.2 112.3 88.0 117.3 106.6 90.9 265.0 236.5 109.1 97.3 89.0 91.9 86.0 93.6 208.0 189.5 85.6 78.0 91.0 132.7 117.3 88.4 274.0 243.2 112.8 100.1 88.7 212.0 200.0 94.3 599.2 561.2 246.5 230.5 93.6
or Total milligrams of inorganic sulfate ethereal sulfate X Total milligrams of inorganic 100% = % inorganic sulfates in urine
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As stated above, one of the advantages of this method is its flexibility. The spectrophotometer, the Leitz clinical photoelectric colorimeter, or even the Duboscq colorimeter may be employed. Separate standard curves must be prepared, however, for each instrument. A standard curve is shown in Figure 2 in which densities, read on the spectrophotometer at 560 millimicrons, are plotted against the concentration (milligrams of sulfate in 7.5 ml. of urine). A standard curve for the Leitz instrument is shown in Figure 3. These solutions were read through the standard green filter No. F 401, supplied by E. Leitz, Inc., New York, N. Y.
Discussion After the solution has been made just acid, the same results are obtained if 0.5 or 1.0 ml. of hydrochloric acid (1 4)is added in excess. The wave length of the filter is not of particular importance since no selective color is being measured; however, wave lengths have been selected to which the eye is sensitive. If aqueous barium chloride is used instead of 20- to 30-mesh crystalline barium chloride, the suspension is not lasting and very low and inconsistent results are obtained. The results on urine samples obtained by the gravimetric method were compared with those obtained by the photometric method, the spectrophotometer being employed to measure densities (Table I). Figures for amounts and ratios were calculated on a basis of 100 ml. of normal urines. Although in some instances the amounts are not in good agreement, the ratios are.
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ANALYTICAL EDITION
SENSITIVITY. A difference of 0.25’ in the spectrophotometric readings corresponds t o about 0.25 mg. of sulfate in 7.5 ml. of urine. I n the case of the neutral wedge photometer, a difference of 1 unit (1 mm.) corresponds to about 0.33 mg. of sulfate in 7.5 ml. of urine. Both instruments are calibrated for slightly finer divisions, but the readings given above may be duplicated without difficulty. Although determinations may be made over a greater range with the Leitz instrument, the sensitivity is little more than 1 mg. of sulfate in 7.5 ml. of urine for a difference of 1 unit. PRACTICAL APPLICATION. The method works well in practice. The urine sulfates of rabbits treated with cyclic compounds were determined by the photometric method and the proportion of the inorganic sulfates was found to have fallen to 10 per cent or less. Upon cessation of the treatment, the inorganic sulfates returned to a normal of 85 to 95 per cent.
The presence of proteins does not interfere measurably with the method, since a solution containing 15 mg. of sulfate and 0.125 ml. of monkey blood serum yielded 15 mg. of sulfate.
Acknowledgment The writers are indebted to W. Deichmann for carrying out the gravimetric sulfate determinations.
Literature Cited (1) C h o l a k , J., IND. ENQ.CHEM., ANAL. ED., 7, 287 (1935). (2) Medes, Grace, and Stavers, Elizabeth, J. Lab. Clin. Med., 25, 624 (1940). (3) Society of D y e r s and Colourists, “Colour I n d e x ” , 1 s t ed., p. 99, Bradford, E n g l a n d , 1924. (4) T r e o n , J. F.,and Crutchfield, W. E., Jr., in preparation.
Ceric Sulfate in the Determination of Iron Using the Molybdisilicic (Silicomolybdic) Acid Methog ALBERT C. TITUS
T
AND
CLAUDE W. SILL, University of Utah, Salt Lake City, Utah
H E authors have varied their method for the determination of iron (3) by substituting ceric sulfate for potassium dichromate, and have found that o-phenanthroline ferrous ion indicator is satisfactory in place of N-phenylanthranilic acid in the ceric sulfate runs. The method and general procedure for runs, blanks, and standardizations follow the ideas presented in the earlier paper. The “stock ferric chloride” and “stock dichromate” were those used in the previous paper. The normality of 0.09733 for the former was taken from that paper. For the normality of the dichromate 0.10058 was used in place of 0.10073 being obtained from pure dichromate from volume ratios of the respective solutions used in titrating the stock ferric chloride by the molybdisilicic acid method. This method of substitution allows cancellation of errors in the procedure and blanks, giving a n “absolute” normality. From the dichromate the ceric sulfate Kas standardized by another substitution procedure, cancelling out any slight errors (1, 4). The oxidizing solutions were in turn run into potassium iodide and acidified with sulfuric acid, and the iodine liberated was titrated with a sodium thiosulfate solution. From the relative volumes of the latter the normality of the ceric sulfate was obtained, after making a suitable correction for acidity differences. The ceric sulfate was also standardized against sublimed arsenic trioxide, using osmium tetroxide catalyst and ophenanthroline ferrous indicator. This method gave a normality of 0.09664 for the ceric sulfate, which is somewhat lower than the values of 0.09704 and 0.09697 obtained by the molybdisilicic (suggested by ill. B. Ptlellon as a name preferable to silicomolybdic) acid method a t the end and beginning of the work, using the stock ferric chloride whose stated normality was shown in the preceding paper to be absolute and so independent of the method used there. However, the thiosulfate method gave 0.09682 for the cerio sulfate normality. It appears that after application of the blanks the molybdisilicic acid method with ceric sulfate gives slightly too low results for iron, since the volume of ceric sulfate used seemed t o be as much as 3 parts in a thousand too low, giving too high a normality above. I n Table I are comparisons of the volumes of ceric sulfate used for the titration of iron, with different indicator combinations. Unless otherwise noted, each figure represents the
average of four runs from which no run varies by over 0.02 ml. I n untabulated cases the blanks were positive but not over 0.02 ml. It will be noted that nonapplication of the blanks resulted in much better agreement than did application. The blanks for the molybdisilicic acid method and the volumes tabulated are those used in calculating the normalities given above. Had the blanks not been applied for the runs a t the beginning of the work the normality would have been 0.09684 in place of 0.09697, a better agreement with the other methods. There is some question as to the real significance of the blanks, especially since their application in the earlier paper resulted in slightly low values for iron as compared with the value by weight. The use of o-phenanthroline ferrous indicator appears to be most satisfactory (Table I). TABLEI. TITRATION OF STOCK FERRIC CHLORIDE WITH CERIC SULFATEO Method
Ceric Sulfate per 45.00 M1. of FeCh
M1. Molybdisilicic acid, Xphenylanthranilic acid (first runs) Same (at end of work)
45.23 45.23
Applied Experimental Blank M1. -0.06 -0.07
Corrected Volume
-0.08 -0.08
45.14 45.13
45.22 45.21 45.23 45.22 45.23C 45.20
ME. 45.17 45.16
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Mercuric chlorides, 0Less than phenanthroline, cold -0.02 hlolybdisilicic acid, o-0.02 45.21 phenanthroline (same -0.02 45.18 directions as with N phenylanthranilic acid) a 32 ml. of 1 t o 1 H&OI added in all runs, in addition t o equivalent of 8 ml. present in added ceric sulfate volume. b N o trouble (8) experienced with adsorption of indicator on calomel with small excess of stannous chloride used. C Three runs only.
Literature Cited (1) F u r m a n and Wallace, J . Am. Chem. SOC.,53, 1283 (1931). (2) L a n g ( p r i v a t e communication cited), Oesper, “Newer M e t h o d s of Volumetric Analysis”, p. 191, N e w York, V a n N o s t r a n d Co., 1938. (3) T i t u s a n d Sill, IND. ENQ.CHEM., ANAL. ED., 13,416 (1941). (4) Vosburgh, J. Am. Chem. SOC.,44, 2130 (1922).
CONTRIBUTION 68 from the Chemical Laboratories of the University of Utah.
