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, u i t h 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 a t 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.
1859
V O L U M E 2 4 , NO. 11, NOVEMBER 1 9 5 2 unsuitable end points, irrespective of solvent ratios. However, Fritz ( 4 ) has successfully titrated caffeine as a base potentiometrically.
tion of the perchlorate salt of theobromine, materially aiding in the viewing of the correct end point. LITER4TCRE CITED
IhTERFEREYCES
Inteifering substances include other bases and water. Most common tablet excipients are not basic and hence do not interfere. DISCUSSION
Theobromine is amphoteric in nature. Recently, Fritz and Vespe (.5) proposed to determine it as an acid. Hoaevei, many tablets containing theobromine also contain other a c t h e ingredients, some of them acidic in character. The proposed procedure circumvents this difficulty. The addition of carhon tetrachloride promotes the precipita-
(1) Conant, J. B., Hall, S . F., aiid Werner, T . H., J . A m . Chent Soc., 49, 3047, 3062 (1927); 50, 2367 (1928); 52, 4-130. 5 1 1 5
(1930). S..-%s%L. CHEY..22. 578 (1950). . . i3j Ibid.,'p. 1028. (4)Ibid., p. 1029. (5) Fritz, J. S.,and Vespe, I-.,.J. A m , Pharni. Assoc.. Sci. Ed., 41, 197 (1952). (6) Sadeau, G. F., aiid Branchen, L. E., J . Am. C h e w SOC., 57, 1336 (1935). ( i )Pifer, C. W., and IT'cllish, E. G., ASAL. CHEM.,24, 300 (1952). (8) Wileon, H. h-.,J . SOC.C h o n . I d . (London), 67, 237 ilO4S). 12) Fritz. J.
RECEIVED for review M a y 29, 1952. Accepted August 2 5 , 1952.
Quantitative Estimation of Total Bilirubin in Serum J4I)IES J. QUIGLEY Diriuion of Laboratories and Research, Y e w Y o r k S t a t e Department of Health, 'ilbany, N. Y .
iM -4s'-
workers hare endeavored t o find a method for estimating the total bilirubin content of serum in a medium in which the protein remains soluble. M o s t of the earlier textbook methods required the addition of ammonium sulfate for precipitation of protein followed by 95% alcohol for solution of the bilirubin. Malloy and Evelyn (6) describe a method, employing a photoelectric colorimeter, for the estimation of bilirubin in serum in which the precipitation of protein and consequent loss of bilirubin has been eliminated. This is accomplished by diluting the serum 1 t o 10 with water and adding absolute methanol t o a final concentration of 50%. A4dlerand Strauss (1, 3) found that caffein, urea, and certain other substances facilitate the coupling reaction between the diazo reagent and biliruliin. Powell ( 7 ) noted that a mixture of sodium benzoate and urea aided in t h e coupling of the diazo reagent and bilirubin and also that the proteins remained soluble. I n his study he used a KlettSummerson photoelectric colorimeter which employs color filters. However, he suggested that the method could be adapted to other photoelectric instruments. Maclay ( 4 ) extended Powell's work for use with a Coleman spectrophotometer and in the preparation of bilirubin standards. However, t h e use of large volumes of reagents makes t h e method cumbersome. The following procedure is an extension and simplification of Powell's and Maclay's methods. REAGENTS
Solution A. One gram of sulfanilic acid (Baker's c.P.) is dissolved in a siiiall amount of water containing 15 ml. of concentrated hydrochloric acid and diluted t o 1 liter xvith distilled water. Solution B. Five-tenths gram of sodium nitrite (1Ierck reagent) is dissolved in 100 m]. of water. Freshly prepared. Solution C. A mixture is also freshly prepared by adding 0.3 ml. of solution B to 10 ml. of solution A. Sodium Benzoate-Urea Solution. Fifty grams of sodium benzoate (llallinckrodt, V.S.P.) are dissolved in 200 ml. of water, warmed if necessary. Fift,v grams of urea are dissolved separately in 200 ml. of water. The two solutions are mixed, diluted t o 1000 ml. with water, and filtered if necessary. The final solution should be Tvater clear. For the diazo blank, solution A is used. Sodium Carbonate Solution. Anhydrous sodium carbonate, 0.75 gram, is dissolved in 100 ml. of water.
50 ml. with chloroform. \\lien not in use, these solutions were kept cold in the dark. Into six test tubes (19 h). 150 nini.) are added respectively 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 ml. of the working solution, V'. This is carefully evaporated, one tube a t a time, with constant shaking in a beaker of hot ivater (80" to 90" C.) until all the chloroform has evolved. Chloroform is poisonous but not flammable. Then 1 ml. of t,he sodium carbonate solution is added to each tube, which is lvarmed to effect complete solution of the residue. The tuhes are cooled in water. Then 1 ml. of a Seitz-filtered serum pool, 1 ml. of solution C, and 7 ml. of sodium benzoate-urea solution are added and well mixed, and 10 to 15 minutes are allowed for full development of color. The final pH should be about 6.0 to 6.5. I n the Coleman spectrophotonieter using 12 X i 5 mm. cuvettes and a wave length of 530 nipfound to be maximal-the percentage transmittance or optical density is read. -4serum reagent blank is made from the same solutions in the same volumes except that solution .\ is substituted for solution C. Five different serum pools, containing negligible amounts of bilirubin were used, as it is imprartiralile to attempt to obtain bilirubin-free serum. While this study was originally aimed a t attaining a rapid, roughly quantitative estimation of bilirubin in serum, the results obtained were more precise than expected. Table I gives the observed transmittance values of the serum pools and of seruin with added bilirubin. Table I1 records optical density data on the same samples. There is a statistically significant trend towards an increasing ratio of 3 to C as C increases-that Is, Beer's law is inadequate t o describe the relationship. However, by the use of either transmittance or optical density data, a curve or table could be prepared suitable for the routine estimation of bilirubin in serum. For the transmittance TITO semilogarithmic paper is used, whereas, for optical density, 0 , the conventional cross-section paper is more suitable.
Table 1. Transmittance T Observed for Given Bilirichin Content Bilirubin Added. Ca
Blank 0.85 o ( T ~ )0 . 8 4 1 0 77
a
PROCEDURE
Coleman's spectrophotometer (Model 6A) was used for the measurements in this study. 9 solution containing 10 nig. of bilirubin (Pfansteil) in 100 ml. of redistilled chloroforn~ was prepared as a stock solution. The working solution, W , prepared as needed, was 10 ml. of the stock solution diluted t o
1
3 6 8 10 a
0.655 0.50 0.37 0.27 0.19
Experiinent Number 7 3 4 Transmittance 0 85 0 825
0.655 0.49 0.37 0 26 0.20
0.85 o 82 0.743 0.635 0.49 0.36 0 27 0.19
5
0.81
0.82
o
0.80 0 73 0.64
0.78 ... 63 0.475 0.25 0.19
0.48 0.34 0.26 0.19
Mean
T ITO
0:8i3 0.738 0.643 0.487 0.360 0.262 0.192
1'000 0.920 o 791 0.599 0 343 0.322 0 236
C is bilirubin added in milligrams per 100 nil. of serum: but only 1 111
of serum was used.
