Coulometric Determination of Selenium KEITH ROWLEY and ERNEST H. SWIFT California lnrtitote of Technology, Pasadena
4, Calif.
T h e coulometric t i t r a t i o n of thiosulfate w i t h iodine has been applied to the determination of microgram q u a n t i t i e s of selenious acid. Two m e t h o d s utilizing reactions used i n conventional volumetric determinations have been studied. Selenopentathionate is formed i n one a n d elemental selenium i n the other. T h e effect of oxygen h a s been investigated. The results obtained f r o m the use of a simple modification of the %orris a n d F a y volumetric t i t r a t i o n of selenium a r e also reported.
.iccordingly, an invePtigat,ion xvas made of the volumetric titration of macro quantities of selenium by two modifications of 1IcNulty’s methotl. In one of these, the selenious acid m s titrated direct,ly with thiosulfate to within a few per cent of the equivalence point before addition of iodide. In the second procedure, the equivalents of iodide added before beginning the titration were small compared with the equivalents of selenium present. These procedures are described below and a discussion is given of the data r e d t i i i g from their use.
T
HE purpose of this work was to investigate the application of the coulometric titration of thiosulfate with electrolytically generated iodine (14) to the determination of microgram quantities of selenium. In preparation for the coulometric titrations a study was made of the modification suggested by McNulty ( 6 ) of the Coleman-McCroskey (1) volumetric procedure. The results of this volumetric study are presented and discussed first, as the same reactions are involved in the coulometric titrations. VOLUMETRIC TITRATION OF SELENIOUS ACID
Two methods for the determination of selenious acid make use of standard thiosulfate solutions (1, 5-6, 9-11, 15, 16). In the first, a large excess of iodide is added, and the iodine is liberated by the reaction
4H+
+ 41- + HzSeOa = Se + 218 + 3H20
(1)
and is titrated with standard thiosulfate (9, 9, 10). This method tends to give low results ( 2 , 9) and especially with other than small quantities of selenium the starch end point is obscured by the red precipitate. In the second method, originated by Norris and Fay (11), an excess of standard thiosulfate is added t o the selenious acid whereupon selenopentathionate and tetrathionate are formed by the folloli ing reaction 4H+
+ 4SpOa-- + HzSeOa = Se(S203)2-- + &OB-- + 3H20 (2)
The excem of thiosulfate is back-titrated with a standard iodine solution. This method has been shown by Coleman and McCroskey ( 1 ) to give results accurate to within 0.2% a t room temperature if a large excess of standard thiosulfate is avoided. In both methods 1 mole of selenious acid is equivalent to 4 moles of thiosulfate. McNulty ( 6 , 7 ) has suggested a combination of these tivo methods in a direct titration. A small amount of potassium iodide is added to the selenious acid and a direct titration is made immediately to the starch end point with standard thiosulfate. Under these conditions the reaction of thiosulfate with selenious acid is faster than its reaction with iodine, therefore Reaction 2 predominates throughout the titration. If starch is present, the starch-iodine color which forms on addition of the iodide is visible throughout the titration; a t the end point, when no significant quantity of selenious acid is left, the thiosulfate reduces the iodine. Under the conditions of the titration only a small amount of selenium is formed and the end point is not obscured. Although McNulty reduced the quantity of added iodide, it was more than equivalent to the selenium present. Thus, unless the titration were made immediately Reaction 1 could predominate; also, his data are inadequate to show the potential accuracv of his method, especially when it is uced vith larger quantities of selenium.
Experimental. CHEMICALS. A stock solution of selenium dioxide was prepared in the following manner. Eight grams of a technical grade of metallic. selenium were dissolved in concentrated nitric acid, and the nitric acid was evaporated. The resulting tetraposit.ive selenium was twice distilled from a concentrated hydrobromic acid solution in order to separate the selenium from tellurium ttnd other elements which are not volatile under these ronditions ( I d ) . Sulfur dioxide was then passed through the distillate until there was no further precipitation. The resulting elemental selenium was filtered and again dissolved in concentrated nitric acid. The nitric acid was evaporated, and the resulting selenium dioxide was twice sublimed. This purified selenium dioxide waa dissolved in a liter of boiled distilled water and standardized according to the method of Coleman and McCroskey (1). Sodium thiosulfate solutions 0.1 V F (volume formal, formula weights per liter, concentrations are used to avoid the uncertainty in the assignment of a normal concentration to thiosulfate used for Reaction 2) were standardized against potassium iodate. Dilute standard solutions of selenious acid and thiosulfate were prepared by appropriate dilution of the above stock solutions. All other chemicals used were reagent grade. TITRATION PROCEDCREB. The two procedures VOLUMETRIC described below were used. Procedure A is probably more accurate but requires a knowledge of the approximate position of the end point. Procedure A. The desired volume of selenious acid solut,ion was pipetted into an Erlenmeyer flask, 5 ml. of 6VF hydrochloric acid were added, and the volume was adjusted to approximatgly 30 ml. This solution was titrated with thiosulfate to wit.hin about 1 ml. of the end point. Then 5 ml. of starch indicator solution and 0.5 to 1 ml. of 1.0VF potassium iodide were added, and the titration was continued to a starch end point. When this procedure is followed, only a slight amount, if any, elemental selenium is apparent a t the end point. Procedure B. After pipetting the desired volume of selenious acid into the flask and adding the hydrochloric acid and water, 5 ml. of starch indicator solution and 2 drops (approximately 0.05 ml.) of 1.OVF potassium iodide were added. The solution was then t.itrated with thiosulfate. When the blue color of the starch iodine complex disappeared, two drops more of 1.OVF potassium iodide were added, and, if t,he color reappeared, the titration was continued until a permanent end point was reached.
Discussion of Results. The experimental results obtained with Procedures A and B are shown in Table I. The quantities of selenium taken are calculated on the basis of the standardization of the selenious acid solution by the method of Coleman and McCroskey ( 1 ) . The agreement is within the volumetric error for both procedures. In outlining Procedure B, it was stated that before the end point was reached, the starch-iodine color disappeared. If the solution was then allowed to stand, the color would gradually reappear. More iodide was added in order to save time. Although Procedures A and B yield comparable results, Procedure A is probably more accurate, as the results become low if Procedure B is modified by adding more iodide initially. When 1 meq. of iodide was added to 2.9 meq. of selenium, the error was -0.7%. When 2 meq. of iodide were added initially to the same quantitv of selenium, the error increases to -1.1%. Thus, the amount of iodide added should be kept as small as possible. 818
V O L U M E 2 7 , N O . 5, M A Y 1 9 5 5
819
Table I. Direct Volumetric Titration of Selenious ;icid with Thiosulfate to the Starch End Point Procedure 4
Taken 141 6
Error
3 537
141 6 141 6 56 72 56 67 3 540
0 0 -0 -0 0
141 6 56 76 3 537
141 7 50 72 3 538
-0 04 0 001
56 76
B
Selenium, Rlg. Found
0 0 04 05 003
01’
Error 70
0 0 0 0 - 0 07 -n 16 0 05 0 07 -0 07 0 03
concentrations of hydrochloric arid and potassium iodide n-ere each 0.1VF. Discussion of Results. The results of determinations by the above procedures are shown in Tables I1 and 111. With tlie exception of the 14 y quantities, all errors are positive and are consistently larger when elemental selenium is formed (Procedure D) than when i t is absent (Procedure C). The per cent error is roughly constant for both procedures. The observation of positive errors in titrations in which elemental selenium was formed \vas unexpected. 7ince negative errors arc observed in Fiiiiilar niarro volunieti,ic tit,rations (2. ! I ) .
COULOMETRIC DETERMINATION OF MICROGRAM QUANTITIES OF SELENIUiM
The coulometric titration of millinormal thiosulfate solutions in O.1VF hydrochloric acid ( 1 4 ) suggested the possibilitj that microgram quaritities of belenious acid might be deteimined by adding an excess of standard millinormal thiosulfate solution and titrating the e\ceas n ith electrolytically generated iodine. Two procedures have been studied. I n one the thiosulfate is added to the selenious acid solution before addition of iodide whereupon selenopentathionate is formed according to Reaction 2. In the other the iodide is added before the thiosulfate, and selenium and iodine are formed according to Reaction 1; the thiosulfate then reduces the iodine. Quantities of selenium ranging from 14 to 1400 y have been determined by both of these methods and the results are shown below. The end points were determined amperometrically. Experimental. APPARATUS. The coulometric and amperometric apparatus has been dexribed (8, IS). The generation equivalent per rates used were 1.037 X lo-’ and 1.036 X second. A galvanometer the sensitivity of which vias set to 0.5 Ha. per division by appropriate shunting was used in measurin the indicator currents. A potential of 200 mv. Tias applie! between the platinum indicator electrodes. COULOMETRIC TITRATION PROCEDURES. Two procedures, C and D, were used. I n both procedures 10 ml. of selenious acid solution and 5 ml. of 1.OVF hydrochloiic acid were pipetted into a titration cell, followed h y 20 ml. of distilled water. The order of addition of thiosulfate and iodide differed in the tmo procedures. I n Procedure C, 10 ml. of dilute standard thiosulfate solution of appropriate concentration were next pipetted into the titration cell followed by 5 ml. of 1.0VF potassium iodide: immediately thereafter the excess thiosulfate was titrated coulometrically with iodine. Selenopentathionate is formed in this procedure. I n Procedure D, 5 ml. of 1.OVF potassium iodide were next added to the titration cell, followed by the thiosulfate solution, and the titration was then made. Elemental selenium is formed in this procedure. Blank thiosulfate titrations were run on solutions which were of similar composition, except that the selenious acid was replaced with 10 ml. of distilled water. With the exception of the blanks for the determination of 1400 y quantities of selenium, these blank thiosulfate titrations agreed within 0.1 % with those calculated from the volume and normality of the dilute thiosulfate solution added and from a blank run on the potassium iodide and hydrochloric acid solutions alone. When blank titrations for 1400 y quantities of selenium were made, a 1% excess of iodine R-as required. This error is attributed to decomposition of thiosulfate to sulfite prior to titration, since a t the beginning of these blank titrations the thiosulfate is 0.002VF and similar errors have been found in previous experiments (14) with solutions having approximately the same thiosulfate and acid concentrations. When 1400 y quantities of selenium were being determined, the excess of thiosulfate was only about one third of the total quantity added, and since the iodine generation time for a selenite titration was only about one third of that for a blank titration, the decomposition of thiosulfate, and, therefore, the error during such a selenite titration should be much less than that during a blank titration. Therefore, for such quantities of selenium, blanks were run on the potassium iodide and hydrochloric acid, and the equivalents of thiosulfate added were calculated on the basis of the dilution of the standard stock solution of sodium thiosulfate. That larger errors were not observed when these quantities of selenium were determined is possibly due to a cancellation of errors. In all titrations and blanks the final volume was 50 ml., and the
Table 11. Selenopentathionate Formed in Coulometric Determination of Selenious Acid Using Procedure C Selenium, y
No. of Titiations
5
Taken 1415 283 0 100.1 141.3 14.11
5 5 7
3
round
Error
Oxygen Not Excluded 1418 3 283 8 0 8 1liO.l 0.0 141.4 0.1 14.07 0 04
lIean
deviation 0.6 0 1 0 1 0 1 0.04
Oxygen Excluded from All Solutions _____ __- 0 4 0 3 283 2 283 6
3
Erior pc 0 2 0 3 0 0 0.1 -0.3 0 1
Tahle 111. Elemental Selenium Formed in Coulometric Determination of Selenious Acid Using Procedure D SPlenirini.
No. of Titration8 3 4
round
Error
Oxygen Not Excluded 1423 8 28.5, 1 2.1 lii0 5 0-1 142.0 0.7 14.07 -0.04
deviation
E1:or.
Fc
0 9 0 1 0 1 0 1 0.0
0 5 -0 8
OxygPn Excluded from All Solutions 283.2 283.6 0.4 0.2
0 1
1-115
?83.0
5
160.1
5 3
i41 3 1.1 11
5
y
Mean Taken
0 ”
0
,
0 3
The consistency of the positive errors suggests that there is an induced ovygen error. In order to check this possibility, titrations were made by Procedures C and D under oxygen-free conditions. All solutions nere prepared oxygen-free by s~ eeping the nater from which they nere prepared with nitrogen. The titrations were then carried out under an atmosphere of carbon dioxide. The results of theqe determinations are shown a t tlie bottoms of Tables I1 and 111. The error resulting from both methods is decreased to about 0.1%; this indicates that the error is due to oxygen. Procedure C, in N hich selenopentathionate is formed, is subject to a smaller ovygen error than is Procedure D ; thereforeits use is recommended. Effect of pH. The coulometric determination of selenium was attempted a t only one other pH value. When the solution was buffered a t a pH of 3, there was no indication of any reaction between the thiosulfate and the selenious acid in the time required for a titration; the same number of reducing equivalents were found as were found in a titration of the thiosulfate alone. ACKNOWLEDGMENT
Fred C. Anson has rendered valuable aid in carrying out certain of the coulometric titrations. Keith Rowley is indebted to the Doiv Chemical Co. for a fellowship for the academic year 10521953 and to the Du Pont Co. for summer research assistance. LITERATURE CITED
(1) Coleman, W. C.. and IIrCroskey, C . R., ISD. ENG.CHEM.,
AKAL.ED..9 , 4 3 1 (1937).
ANALYTICAL CHEMISTRY Gooch, F. A., and Reynolds, W. G., Z . araorg. Chem., 10, 248 (1895).
Klason, P.. and SIellquist, H.. 2. angew. Chem., 25,514 (1912). Klein, A. K.. J. Assoc. Oflic. d g r . Chemists, 24, 363 (1941). 3IcCullough, J. D., Campbell. T. IT.,and Krilanovich, S . J., A s ~ L CHEX., . 18, 638 (19461. llcru'ulty, J. S., Ibid., 19, 809 (1947). NcNulty, J. S., Center, E. J., and JIacIntosh, It. M.,IbLd., 23. 123 (1951). Meier, D. J., Myers, R. J., and Swift, E. H., J . Am. Chem. Soc., 71, 2340 (1949).
JIoser, L., and Prim, W., 2. anal. Chem., 57, 277 (1918). lluthman, W.. and Schaefer, W., Ber., 26, 1008 (1893).
(11) Sorri-. J. F.. and Fay, H., Am. Chern. J . , 18, 705 (1896). (12) Noyes.. .1.A , and Bray, W. C., "Qualit,ative Analysis for the Hare Elements," p. 37, Jlacmillan, New York, 1927. (13) Rainsey, W. J., Farrington, P. S . , and Swift, E. H., - 4 s . i ~ . CHIX., 22,332 (1950). (14) Howley. K., and Swift, E. H., Ibid.. 26,373 (1954). (15, van der Meulen. J. H.. Cheni. Weekblad. 31. 333 (1934). (16) Werniniont, G., and Hopkinson, F. J., IND. ENG.CHEM, Asar. ED.. 12, 308 (1940). I~
RECEIVEDfor review Septeiiiher 2 . 1934. Accepted October '23, 1934. Contribution No. 1935 from the Gates and Crellin Laboratories of Clieniistry. California Institute of Teclinology, Pasadeua 4, Calif.
Chromatographic Separation and Determination of Porphyrin Methyl Esters D. A. RAPPOPORT, C. R. CALVERT, R. K. LOEFFLER, and J. H. GAST Departments o f Radiology and Biochemistry, Baylor University College o f Medicine, Houston 25, T e x .
By means of horizontal paper chromatographj with filter paper placed between glass plates, the methyl esters of uroporphyrin 1. coproporphyrins I and 111. and protoporphyrin IX were separated within 1 hour. Elution of individual bands from the paper chromatogram followed b! fluorometric analj ses permitted quantitative estimations of each porphy rin ester.
D
URIXG an investigation of various methods for the separxtion and quantitative determination of porphyrin isomers, difficulties were experienced in separating for analyses sufficient arnounts of various porphyrins from biological fluid laboratory, repeated clear-cut separations of porphy esters from biological fluids with the paper chromatographic niethods of Chu, Green, and Chu ( 2 ) and the modifications of this method by Bogorad arid Granick ( 1 ) \vas found to be difficult hecause of interference by impurities. The resultant bands of the porphyrin esters, from either ascending or descending chromatograms, were somen-hat irregular and overlapping and a t best insufficiently separated for quantitative recoveries. Lysing horizontal paper chromatography with a modified solvent system, a rapid method for the complete separation of methyl esters of uroporphyrin I! coproporphyrin I, coproporphyrin 111, and protoporphyrin I S has been developed which permits quantitative estimation of each of these porphyrin esters.
circle 1 inch in diameter. This circle is divided into arcs by two to a i s radial lines allotving as many as six samples to be chromatographed simultaneously. Developing Solvents. SOLYESTii. Three volumes of petroleum ether (boiling point 30" t o 60" C.) and l volume of chloroform. SOLVEST B. Twenty volumes of heptane, 1 volume of et,hylene dichloride, and 1.5 volumes of tert-but,yl alcohol. AL-XILIARY SOLVEST.Equal volumes of petroleum ether and chloroform. 3Iethyl esters of coproporphyrins I and 111and of uroporphyrin were furnished by Samuel Schn-artz. Paper chromatographic analysis indicated that the esters were single components with only traces of impurit,ies. The methyl ester of protoporphyrin I S was prepared from crude protoporphyrin (H. I f . Chemical Co., Ltd., Santa Monica, Calif.) and purified by means of paper chromnt ography . L-ltraviolet, lamp, 380 nip, Hanovia Type 1C103 (Hanovia Chemical and Mfg. Co., Sen-ark, S . J.). Fluorometer, Lumetron Model 402-EF, used with a priniarifilter of Corning g h s 5513 (365 mp) and secondary filter of CO;-
EQUIPMENT AND MATERIALS
Two glass plates, 13 inches square, cut from 0.25-inch commercial plate glass. Any other suitable size may be used. One glass square is marked a t the exact center with a wax pencil, and a hole 0.25inch in diameter is drilled through. This is done by forming a ridge of putty around the center mark so that water can stand over this area. Using a short length of 0.25-inch brass tubing in an electric drill press and adding abrasive powder intermittently, a hole is drilled halfway through on one side of the plate, then completed by drilling from the opposite side. Two barrels from hypodermic syringes, 10 or 20 ml., each of which is fitted with a piece of thin-walled (5-mm. outside diameter) gum rubber tubing over the entire length of the syringe tip. Each syringe is packed with Whatman cellulose powder to a depth of about 2 cm. Before use, each packed syringe is rinsed with the particular solvent to be used for chromatography, and the rate of solvent flow is adjusted to approximately 1 drop per 2 seconds by tamping the cellulose packing with a glass rod. Six lead bricks, weighing about 25 pounds each (Xuclear Instrument and Chemical Corp., Chicago, Ill.). Filter paper sheets, Whatman No. 3MM, cut in squares (13 X 13 inches) the same size as the glass plates. At the center of each paper the location for the sample is made by drawing a
Figure 1. Schematic representation apparatus used in horizontal paper chromatography
