V O L U M E 2 4 , NO. 11, N O V E M B E R 1 9 5 2 tions of the dye are stable if kept out of contact with air by means of a blanket of nitrogen. The author is unable to offer a theory which would account for all of these phenomena. It is therefore empirically recommended that the indicator be prepared either with diethanolamine or with triethanolamine and that no other solvents or salts be added. There appears to be little choice between diethanolamine and triethanolamine. No advantage is gained by protecting the solutions against light or by keeping them in a refrigerator. The indicator solutions are best dispensed in prescription type dropper bottles, which should be kept tightly closed when not in use to avoid the absorption of moisture by the (hygroscopir) alkanolaminw. ACKNOWLEDGMEhT
The author is indebted to llaurice E. Selson, LouiseC.Schauer, Doroth? Llargolin, and Albert A . I'oithal of this department for testing various indicator solutions under different conditions and reporting result?: and to Joseph J Connors, East Bay Municipal
1857
Utility District, Oakland, Calif., and John D. Betz, Bet2 Laboratories, Philadelphia, Pa., for helpful discussions. LITERATURE CITED
(1) .Im. Pub. Health Assoc. and Am. Water Works Assoc., "Standard Methods for t h e Examination of Water and Sewage,'' 9th ed., p. 23, 1946. (2) Banewicz, J. J., and Kenner. C . T., AXAL.CHEST.,24, 1186-7 (1962). (3) Betz. J. D.. reviewer's comments. . h u . 1. 1952. (4) Betz, J. D., and Koll, C. A., J . A m y Water T o r k s Assoc, 42, 49-56 (1950). (5) Ibid., pp. 749-54. (6) Biedermann, W., and Schwarxenbach, G., Chimza, 2, 56-9 (1948). (7) Connors, J. J., personal communication, Oct. 16, 1951 (8) Hall Laboratories, Inc., Bull. 1 RE 50, 1950. (9) Llarcy, V. M., Power, 94, 105-8 (1950). (10) Porter, 3. D.. Chemzst-Analyst, 41, 33-5 (1952) RECEIVED for review July 7 1932
Accepted August 25, 1952
Preparation of Very Dilute Standard Base by Ion Exchange BESJAMIK WOLF GRUNBAUM, WOLFGANG SCHONIGER~,AND PAUL L. KIRK Diuision of Biochemistry, C;nir.ersity of California Medical School, Berkeley, Calif.
'
iX DEVELOPIXG a method for titration of microgram quanti-
ties of fatty acids from fractions of biopsy specimens of liver, a major problem was the difficulty of obtaining a constant normality of standard alkali of about 0.001 N . Solutions were carefully prepared with boiled, redistilled water and saturated caustic from which the carbonate had been allowed to precipitate. On storage of such solutions in the usual manner in bottles well coated with paraffin, and without contact with glass or rubber, the strength of the solutions was found to differ from day to day and even during the course of the day from storage, handling, or other cause. Schubert (1), describing analytical applications of ion exchange separations, quotas the suggestion of a simple means for preparing standard acid by stoichiometric release of hydrogen ions from organic cation exchange resins. The preparation and storage of very dilute mineral acid is certainly less critical than is that of very dilute alkali, but the suggestion indicated the possibility of a similar preparation of alkali by means of anion exchanging resins to obtain stoichiometric release of hydroxyl ions.
vessel with an outlet about 1 cm. below the top. Solution flowing from this outlet was led to a drain by a rubber catheter tube. To the lower end of the vessel was attached a capillary stopcock, below which was an angular side connection and delivery tip as shown. The side connection was attached by means of a gum rubber tube to the capillary tip of a capillary buret ( 2 )which could be filled after opening the stopcock. When the stopcock was closed, the incoming standard solution was forced out of the outlet and led to the drain.
I"
Since this paper was submitted, Steinbach and Freiser ( 4 )have described a similar technique for preparation of batches of 0.1 N sodium hydroxide with Amberlite IRB-400. It is clear that the technique is of general application but n-ith special advantage in titration of microgram quantities. This idea was satisfactorily developed by means of a simple apparatus in which a strong base anion evchange resin mas reacted with 0.0010 N potassium chloride solution to provide the corresponding standard potassium hydroxide solution according to the reaction
RX-OH-
+ K f + C1- +R S + C l - + K - + OH-
The reaction apparently proceeds without attention for months and shows a remarkable constancy of normality of the solution delivered. EXPERIMENT 4 L
Ap aratus. The apparatus consisted of a glass tube, 40 cm. in lengt! and 16 mm. in outside diameter, attached a t the lower end to a 1-mm. bore heavy-wall capillary tube bent as shown in the diagram. A sintered-glass filter sealed in the larger tube served to retain the resin with which the tube was filled. The constricted end of the capillary was led to the bottom of a reservoir 1
Permanent address, Pregl Laboratories, University of Graz, Austria.
Above the column was placed a separatory funnel containing 0,0010 N potassium chloride solution which was led through a length of Tygon tubing to a tip in the top of the column. Sotches were filed in the stopcock plug to allow fine control of the flow rate which could also be altered by the height of the funnel. Procedure. The column was charged with the strong base anion exchange resin, Amberlite XE-67. The resin has been previously saturated with a 4% sodium hydroxide solution and washed with redistilled water until a neutral reaction was obtained. The separatory funnel filled with 0.0010 N potassium chloride solution was connected and about 200 ml. of solution allowed to flow rapidly through the column to displace water, after which the rate was adjusted to about 2 ml. per hour. The
1858
ANALYTICAL CHEMISTRY
excess alkali generated was allorved to waste to the drain because interference with the rate of flow appeared to alter the normality slightly. The buret was filled after opening the stopcock, during which the delivery tube could be flushed by raising the catheter tube drain. On closing the stopcock and adjusting the solution meniscus in the buret, the titration could be performed, After the system reached equilibrium the only attention required was to replenish the stock of 0.0010 N potassium chloride in the upper separatory funnel from time to time. DISCUSSION OF RESULTS
Sumerous titrations were performed against a known standard acid solution and against potassium biphthalate and benzoic acids as primary standards, with both color indicator and potentiometric end points. -4closed-chamber titration vessel similar to that previously described ( 3 ) was employed. Stirring was performed with a rotating magnetic stirrer. Carbon dioxide that remained in the chamber on closing was absorbed by means of a little saturated sodium hydroxide solution kept in the bottom of the chamber. Over a period of 45 days, the alkali resulting from the ion exchange process was titrated with standard acid using a mixture of bromocresol green and methyl red as indicator. A total of 56 titrations during this period gave an average titer of 43.34 microliters of acid with a maximum deviation of zk0.50 microliter and a standard deviation of =kO.171 microliter. A small positive deviation of normality of the alkali from that of the potassium chloride solution r a s noted. The reason for this i q not clear, and may be related to the difficulties of employing primary standards in minute quantities. Benzoic acid, titrated in alcoholic medium, yielded che most consistent values for absolute Btandardixation, uith a value for the alkali of 0.00105 It had been noted by Sheldon Rosenberg of this laboratory in
connection v ith other investigations that as hydroxyl ion was replaced by chloride ion with this resin, the color of the resin n-as altered noticeably. The color change apparently serves as a reliable index of the degree of exhaustion of the material. The rate of change noted in these experiments indicates that the column described will function with this concentration of solution for a period of at least 8 to 10 months. Using the manufacturrr’s figures that 1 ml. of wet resin --ill exchange 1 milliequivalent of chloride for hydroxide ion, it follou-s that the amount of material used in this experiment should last for a t least 1 year a t the rate of flow employed. The ion exchanger XE-67 is stated t o operate best with dilute solutions-e.g., not over 0.01 N . It appears probable that within this limitation, any normality of alkali solution may be readily obtained aithout interference of carbonate or other ions and over a period long enough t o be convenient as a general technique. ACKNOWLEDGMENT
This investigation was supported by grants from the Veterans Administration, Contract S o . V1001M-1979, and the Research Committee of the University of California. Gratitude is due E. L. Duggan for supplying the hmberlite resin and for his helpful suggestions. LITERATURE CITED
(1) Schubert, J., 4 x . 4 ~CHEM.. . 22, 1359 (1950). (2) Kirk, P. L., “Quantitative Ultramicroanalysis,” p. 31, S e w York, John Wiley & Sons, 1950. (3) Ibid., p. 124. (4) Steinbach, J., and Freiser, H., ASAL. CHEY.,24, 1027 (1952). RECEIVED for review June 17,1952.
Accepted August 25,1952.
Titration of Theobromine in Nonaqueous Solutions .-IRTHUR POULOS Control Laboratories, Winthrop-Steams, Inc., Rensselaer, .V. Y .
HE extensive studies on “super acids” by Conant, Hall, and rWerner ( 1 ) illustrated that many organic bases too weak to be titrated in water could be successfully titrated in glacial acetic acid. The successful titration of amino acids in this medium was the first nonaqueous acid-base titration to enjoy wide acceptance. The success by Nadeau and Branchen (6) gave impetus to this field of study. Wilson ( 8 ) , and later Fritz ( S ) , found that end points could be sharpened through the use of a mixed solvent system such as glacial acetic acid and a hydrocarbon. Fritz ( 2 ) also employed amphiprotic solvents such as p-diosane. Pifer and Wollish (?) succesefully titrated many weak bases potentiometrically. These principles were applied to a procedure for the titration of theobromine, a widely used alkaloid, with emphasis on readily obtained solvents and the use of a colored indicator.
cator solution. The color change is from yellow on the alkaline side to green on the acid side. Theobromine, due to the basic nitrogen in the 9-position, consumes 1 equivalent of perchloric acid. Results are listed in Tables I and 11. SCOPE
Many weak organic bases may be accurately titrated in glacial acetic acid and other nonaqueous solvents. Success with theobromine encouraged attempts with other bases in the purine group. It was found that poor end points were obtained with theophylline using the procedure as set down. By increasing the carbon tetrachloride content to 100 ml. however, a reasonably sharp end point was obtained. All attempts with caffeine gave
Table I. Titration of 0.3-Gram Samples of Pure Theobromine
REAGENTS AND SOLUTIONS
Sample No.
Recovery, To
Glacial acetic acid, U.S. Pharmacopeia. Carbon tetrachloride, Sational Formulary. Theobromine, commercial samples as received. a-Naphtholbenxein indicator, 1% acetic acid solution. Acetous perchloric acid, 0.1 N , prepared by diluting 8.5 ml. of 72% perchloric acid to 1000 ml. with glacial acetic acid. This material is standardized against acid potassium phthalate.
1
99.47 99.79 100.11 99.32 99.67
PROCEDURE
Dissolve 0.3 gram of theobromine in 30 mi. of glacial acetic acid with the aid of gentle heating. Cool to room temperature and add 30 ml. of carbon tetrachloride. Titrate with 0.1 N acetous perchloric acid using 3 drops of or-naphtholbenzein indi-
2 3 4
Av.
Table 11. Titration of 0.3-Gram Samples of Theobromine in Tablets (80% Theobromine) Sample No.
Recovery, To
% of Theory
1 2 3 4
79.60 79.88 79.88 80,04
99.50 99.85 99.85 100.05 99.81
Av.
