Titrimetric Determination of Sulfates by Diazo Titration of Benzidine Sulfate R. E. KELLER and R. H. MUNCH Research Department, Monranto Chemical Co., St. Louis, M o .
S
EVERAL titration methods have been proposed for sulfates which depend upon the titration of precipitated benzidine sulfate. Raschig (7, 8) developed a benzidine precipitation method whereby the precipitated benzidine sulfate is removed by filtration, suspended in water, and titrated with standard alkali a t 50’ C. This method is used extensively for determining sulfur in organic as well as inorgamc substances. I t has the disadvantage of a slo~vand tedious titration resulting from the relativeIy insoluble benzidine sulfate ( I , 6). Poor accuracy is noted with lon sulfate concentrations owing to the slight solubility of benzidine sulfate in aqueous solution. Callan and Toennies ( 1 ) by using a procedure based on one originally published by Fiske (3’reduced this solubility with an acetone-n-ater precipitation medium. Raiziss and Dubin (6) have recommended the direct titration of benzidine sulfate n i t h potasqium permanganate solution, but the end point is not sharp ( 2 ) . This method has been modified by Hibbard ( 4 ) , who recommends the use of a measured excess of potassium permanganate and the titration of the excess n i t h standard oxalic acid. Ollgaard ( 5 )determined sulfates in serum by precipitating with benzidine and by then titrating the benzidine sulfate with standard barium chloride solution, using rhodizonic acid as the indicator. The method described here involves benzidine sulfate precipitated from an alcohol-water solutioh with a subsequent diazotization titration procedure. This investigation was carried out to find a rapid proredure for the determination of sulfates in the presence of certain sulfonates.
Table I.
Determination of Sulfates in Synthetic Sulfonate Samples
Sample SulConcn., fonate gram TS 1.0 1.0
A.4S
0.5 0.5 0.5 0.5 0.5 0.5
BS
Sulfate Added=,
%
Sulfate Found,
%
Error,
ro
0.28 0.29 +2.8 -1.9 1.40 1.37 2.92 -0.1 2.92 8.34 -0.7 8.29 0.60 0.58 -3.0 1.19 1.15 -3.0 2.92 2.92 -0.1 10.75 10.67 -0.8 +0.7 0.60 0.61 2.92 ’ 2.92 -0.1 10.83 +0.8 10.75 Toluene sulfonic acid, c.P.,neutralized with so-
1.0 0.5 0.5 TS. Toluene sulfonate. dium hvdroxide. AAS: Alkyl aryl sulfonate. BS. Benzene sulfonate. Benzene sulfonic acid, practical, neutralized with sodium hydroxide. a Sulfate added as sodium or potassium sulfate.
lead for the pH meter. The solutions are stirred with an airdriven glass stirrer. REAGENTS
Benzidine dihydrochloride. Weigh 10 =k 0.1 grams of reagent grade benzidine dihydrochloride into a 500-ml. volumetric flask, add 50 ml. of distilled water and 2 ml. of concentrated hydrochloric apid, and dilute to volume with distilled water. Potassium nitrite, standardized 0.1M. Potassium bromide, crh stalline. Hydrochloric acid, c.P., 6-J-. Ethyl alcohol, 95%. PROCEDURE
Precipitation. The method can be applied to a wide range of sulfate concentrations by varying the reagent volumes. The proper volumes depend upon the concentration of sulfate in the sample. The sample size aiid reagent volumes used for each are as follows: Sulfate,
70
Sulfate, hlg.
Sample, Gram
Water, MI.
Ethyl Alcohol, M1.
Benzidine Reagent, M1.
The total volume of 100 ml. should not be exceeded for complete benzidine sulfate precipitation. Samples containing more than 12% sulfate are analyzed by using a sample size which will produce 100 to 120 mg. of sulfate ion. Select the proper reagent volumes from the table, Keigh the sample accurately into a beaker, and dissolve it in a minimum of water. Neutralize the solution with either 5.V hydrochloric acid or 5 s sodium hydroxide solution to a phenolphthalein end point and make it acidic with a slight excess of 0.1 -V hydrochloric acid. Dilute to the proper water volume and add the designated volume of ethyl alcohol. Introduce the benzidine dihydrochloride reagent dropwise with stirring and allow the solution to stand 5 to 10 minutes. Titration. Transfer the precipitate from the beaker to a Gooch crucible prepared with an asbestos mat and wash with 50 ml. of ethyl alcohol using gentle suction. Transfer the washed precipitate and the asbestos mat quantitatively to a 150-ml. beaker with 10 ml. of distilled water and add 50 ml. of 6N hydrochloric acid followed by several crystals of potassium bromide. Titrate potentiometrically with standard 0.1M potassium nitrite solution using an air-driven stirrer. Add potassium nitrite solution until approyimately -600 mv. is recorded with a platinum electrode or -500 mv. with a tungsten electrode, then add 0.10-ml. increments every 30 seconds and read 15 seconds after each addition. The end point corresponds to the largest potential change between successive increments. CALCULATIOh
4PPAR 4TUS
The preripitate is collected on a thin asbestos mat in a Gooch crucible with the aid of a crucible holder and suction flask. The electrode potentials are measured with a Leeds and Xorthrup pH indicator, Model 7664-ill. Tungsten (wire)-calomel or platinum (foil)-calomel electrode systems with a sleeve-type saturated potassium chloride salt bridge for the calomel electrode have been used. The platinum electrode coneists of an 8 X 20 mm. piece of platinum foil fused t o a platinum w r e n Ith the wire sealed in the end of a 4-mm. diameter glass tube to provide contact for an electrical lead. The tungsten electrode consists of a tungsten wire (1 inch in length and 0.040 inch in diameter) sealed in the end of an 8-mm. glass tube. il mercury pool serves to make contact between the electrode and the wire
The per cent sulfate can be calculated in terms of sulfuric acid as indicated in the following equation.
RESULTS A N D DISCUSSION
Synthetic mixtures of 0.10 to 12.00% sulfate in toluene sulfonate, benzene sulfonate, and an alkyl aryl sulfonate n-ere prepared with chemically pure reagents. ilnalyses of eleven synthetic samples show good agreement between the experimental data and the actual composition of the samples. Table I gives the data for these investigations. The maximum error for the 1518
V O L U M E 2 6 , NO. 9, S E P T E M B E R 1 9 5 4
1519
Table 11. Reproducibility with Different Variables Analyst A A B B B B B B
Electrode System Pt-calomel Pt-calomel W-calomel W-calomel Pt-calomel Pt-calomel Pt-calomel Pt-calomel
Readin Interval After I d d i t i o n , Sec
NasS01, Mg. 15 75.00 15 37.50 30 75.00 37.50 30 37.50 30 15 37.50 15 37.50 37.50 15 Arithmetical mean Average deviation Average deviation of mean
NarSOc Found,
%
100.07 100.29 100.12 100.43 100.12 100.28 100.12 100.12 100.19% &0.104% 0.085%
0 to 2% sulfate range i i 4~3.0%and for the 2 to 12% sulfate range &08% of the amount added. Several samples of impure toluene sulfonic acid containing 0.0 to 0.90% sulfuric acid have been analyzed. The results agree with gravimetric analysis data with the same degree of accuracy as was obtained with the synthetic samples. Samples of benzene sulfonic acid containing as much as 50% sulfuric acid have been assayed by this procedure. Phosphate, chromate, and chloride ions interfere with most benzidine sulfate precipitation procedures. The effect of these ions and a ferric salt upon the accuracy of the described method has been studied. -4nalyses of synthetic samples containing 50.0 mg. of sulfuric acid and varying concentrations of each ion have shorm that 5 mg. of phosphate (as NazHPOa), 10 mg. of chromate (as K2Cr04),500 mg. of chloride (as XaC1) and 10 mg of ferric chloride will not interfere with this method of analysis. If the concentration of these ions exceeds the stated limits they should be removed or rendered ineffective. I t is suggested t h a t this can be done by the procedures given by Telcher (9). It I S recommended that the titration be follon ed n i t h a tungsten-calomel electrode system. The authors have found that some platinum electrodes give as good results as tungsten, but others give much poorer results. The average potential break a t
the end point with a tungsten-calomel electrode system per 0.1 ml. of 0.1M potassium nitrate solution is 114 mv This figure is obtained from titration data from the analyses of 252 benzene sulfonic acid samples. The largest potential break was 210 mv. and the smallest was 60 mv. The precision of the method was checked under various experimental conditions. Two analysts with separate reagents and equipment analyzed aliquots from a standard solution of sodium sulfate according to the conditions given in Table 11. Good reproducible results were obtained with an average deviation of 50.10%. The accuracy was determined by analyzing a standard solution of 0.2N sulfuric acid. Six samples gave an average of 99.87 i 0.15% sulfuric acid; theoretical value 100.00% sulfuric acid. The method has proved satisfactory for the determination of sulfate in three sulfonates. It can be used for the determination of sulfates in other mixtures with comparable results. The main advantage of this method over conventional gravimetric and volumetric procedures is the short assay time. ACKNOWLEDG3IENT
The authors gratefully acknowledge the help of C. H. Brackbill in obtaining experimental data. LITERATURE CITED
(1)
Callan, T. P., and Toennies, G., IND. ENG.CHEM., =lxua~.ED., 13, 450 (1941).
(2) Dmmmond,'J. C., Biochem. J . , 9, 493 (1915). (3) Fiske, C. H., J . B i d . Chem., 47, 59 (1921). (4) Hibbard, P. L., Soil Sci., 8, 61 (1919). ( 5 ) Ollgaard, E., Biochewz. Z., 274, 181 (1934). (6) Raisiss, G. W., and Dubin, H., J . Bid. Cheni., 18, 297 (1914). (7) Raschig, F., 2. angew. Chem., 16, 617, 818 (1903). (8) Ibzd., 19, 331 (1908). (9) Welcher, F. J., "Organic Analytical Reagents," Tola 11, p. 300, S e w York, D. Van Sostrand Co., 1947. RECEIVED for rpview September 21, 1953
Accepted J u n e 7 , 1954
Infrared Identification in Paper Chromatography T. Y. TORIBARA
and VICTOR DI STEFAN0
University o f Rochester, Rochester,
I
N. Y.,
and M a r q u e t t e University School o f Medicine, Milwaukee 3,
KFRARED spectrophotometry for the study of biological materials, especially those isolated by paper chromatography, has always appeared as an attractive possibility, but previously existing sample-handling techniques have been unsuitable. The quantities of material separated by a paper chromatogram are in the fractional milligram range. The conversion of such minute quantities to a suitable sample for infrared studies has been made possible through the development of the method for making dilutions of organic solids in solid potassium bromide by Stimson and O'Donnell ( 7 ) and Schiedt (4-6) Suitable records were obtained from these small samples by using the beam condensing system of Anderson and Woodall (2) which has provided a simple means for extending the analytical range of the spectrophotometer to samples as small as 10 y. The procedure of freeze drying a solution of the sample and potassium bromide was selected as the best method for quantitatively transferring and uniformly dispersing these small samples Fortuitously, freeze drying has been shown by Schiedt ( 4 ) to give the optimum particle size for the production of the most satisfactory spectral records. Because of the obvious advantages of this method, the procedure as adapted for small samples is described in some detail in the following sections.
Wis.
In-the course of work on the calcification mechanism it wa8 suspected that an organic phosphate compound could be a possible intermediate in the deposition of bone salts. Indeed, such a compound was found in protein hydrolyzate8 of both bone and calcifiable cartilage (3). The two-dimensional chromatogram of L) cartilage hydrolyzate is represented schematically in Figure 1. The unknown spot is the suspected organic phosphate. T o gain some insight into the nature of this unknown material, a sample was isolated for study by infrared analysis. I n order to obtain larger quantities of the unknown substance than would be separated by the usual two dimensional chromatography, the isolation wa3 carried out as two separate single-dimensional steps. ISOL4TION OF SAXfPLE BY PAPER CHROIIATOGRAPHY
Six milligrams of calcifiable rachitic cartilage from the tibia of albino rats were placed in an ampoule containing 5 ml. of 6.V hydrochloric acid. The ampoule was sealed and placed in boiling nat'er for 21 hours. The hydrolyzate was filt'ered, evaporated to dryness, and reconstituted with distilled mater. This process was repeated once again to remove the hydrochloric acid. Finally, the residue was dissolved in a convenient quantity of distilled water (usually 1 ml.) and transferred by means of a
