Determination of Carbonyl Grouns 1
in Bile Preparations
ARIOS E. LIGHT The Burroughs Wellcome 8i C o . U. S. A. Experimental Research Laboratories, Tuckahoe, N. Y.
E
XPERIRIEXTAL evidence has been obtained recently
should be added at the start of the reaction when there are large amounts of keto groups present in the unknowns, SO that approxiprecipitate may be obmately the same quantity Of the tained in each reaction. Therefore, in testing recoveriesllwith 0.05-gram samples of triketocholanic acid (made by dissolving 0.5 gram of pure triketocholanic acid in 1 cc. of sodium carbonate solution and diluting to 10 cc.; 1 cc. contains the 0.05-gram sample needed), 3 cc. of the amine solution (0.0781 gram or 9 mole equivalents to react with the mole equivalents of the keto groups) are pipetted into the test tubes. The unknowns, which in the following cases are low inrketo groups, are similar to the controls except for the addition of 0.03
FThich suggests that carbonyl determine the choleretic activity of bile salts used in gall bladder therapy (1). Various methods have been presented for the quantitative determination of these groups in different types of chemical compounds but ferv have been applied to the bile constituents. This article presents a comparison of the usefulness of two methods, both of which are based on the reaction between the keto groups and hydroxylamine with the resultant oxime format ion.
TABLE I. COMPARATIVE ANALYSES ---Gravimetric MethodC=O, Standard K O . of deter70 deviation minations 21.7 1.87 12 20.9 1.6 2.02 6 -n..i 6 0.9 1.66 4.0 7 25.88 3.98 9 26.45
Preparation Triketocholanio acid" Theoretical Ox bileb Turtle bileb Pig bileb Godium pyruvate Theoretical .... Acetone Theoretical a Decholin, Riedel-de Haen, Inc., New York, N . Y. b Burroughs Wellcome & Co. (U.S. A.) Inc., New York. S . Y. c Pigment masks indicator.
..
--
Titrimetric Method Indicator Standard No. of deterC=O, deviation minations % 0.146 7 19.92
.
C=O,
% 20.22
6
OC
..
2.24
0:344
...
..
47.3s 48.23
0.057
G
...
6
7 -
0 0 0
-
Glass Electrode-Standard No. of deterdeviation minationr 0.335
...
...
45.99
1.92
gram of the bile preparation. Each sample is run in duplicate Water is now added to some to equalize the volumes, 0.5 cc. of sodium carbonate solution is added to each, and the tubes are stoppered tightly with rubber stoppers. After a thorough mixing of the contents the tubes are heated in water at 80" C. on a steam bath for 2 hours. Allowing the mixture to stand at room temperature overnight and then heating for a shorter time is equally effective. The oxime of the cholanic acid usually precipitates as the reaction proceeds. After cooling, the tubes are opened, stoppers washed, and the contents acidified with concentrated acetic acid. (Alkacid test ribbon, Fisher Scientific Co., Pittsburgh, Penna., is very convenient.) Ethyl alcohol is used to prevent foaming. When precipitates are present they are filtered off and washed three times with small amounts of dilute acetic acid. The solutions of all are transferred with washing to 30-cc. beakers and should at this point approximate 10 cc. in volume. They are made distinctly alkaline with concentrated ammonium hydroxide, and 1 cc. of diacetylmonoxime (0.1516 gram or 12 mole equivalents) is added to each. The color should be slightly yellon; if not, more ammonium hydroxide should be added. The solutions, covered with watch glasses, are now heated for 2 hours on a steam bath at 80" C. Crystals of dimethylglyoxime which appear at this stage and increase in amount if the solutions are allowed to cool are redissolved by adding 5 cc. of alcohol to each sample. Into each beaker is pipetted 0.75 cc. of the nickel acetate solution (0.14 gram or 9 mole equivalents) and the solutions are digested on the steam bath for 2 hours longer Rithout covers but with frequent stirring. After cooling or standing overnight each solution is transferred to a tared 14-cc. conical, heavy-walled Pyrex centrifuge tube by means of a rubber policeman, with addition of ethyl alcohol (to prevent creeping) and dilute ammonium hydroxide. After centrifuging, the red precipitate is suspended in 6 cc. of dilute ammonium hydroxide, centrifuged again, and dried to constant weight at 110" C. A very thin layer of alcohol in the tubes permits manipulation without appreciable loss of ma__ terisl.
The titration method of Bryant and Smith (S), which determines the hydrochloric acid liberated from the amine salt after the oxime formation Ivith pure aldehydes and ketones, was found satisfactory for bile preparations which contained no pigments to mask the indicator employed. This limitation was overcome by electrometric titration. Gustus (unpublished data reported by Ivy et al., 1 ) determined keto groups in bile preparations by a gravimetric method originally indicated b y Hirschel and Verhoeff (4) which is based on the recovery of excess hydroxylamine. No details were given but the procedure was described as lengthy and tedious. The method herein discussed also entails the reaction with diacetylmonoxime to form dimethylglyoxime and its subsequent precipitation as the nickel salt of dimethylglyoxime. Under the conditions given below fairly accurate values for carbonyl groups in bile preparations can be obtained in a reasonable length of time.
Reagents for Gravimetric Method Hydroxylamine hydrochloride, anhydrous c. P., 0.6513 gram in 25 cc. of aqueous solution. Sodium carbonate, anhydrous c . P., 13.25 grams in 100 cr. of aqueous solution. Nickel acetate tetrahydrate, c. P., 4.665 grams in 25 cc. of aqueous solution. Diacetylmonoxime, Eastman Kodak Co., 3.79 grams in 25 cc. of aqueous solution (heat). Ethyl alcohol, 95 per cent. Ammonium hydroxide, concentrated and dilute. Acetic acid, concentrated and dilute.
Procedure
Calculations
The first reaction is carried out in 15 X 125 mm. Pyrex test tubes. Since the conditions of each run determine the recovery Of the amine are carried of hydroxylamine, two along simultaneously with the other ten or more samples in a single batch determination. These controls contain 2 CC. or 0.0521 gram of the amine hydrochloride. Larger quantities
The weight of the nickel dimethylglyoxime multiplied by tile factor 0.4812 equlils the equivalent weight of hydroxyla m h e hydrochloride. The recovery factor, usually over 90 per cent, is determined by dividing the weight of the recovered 42
ANALYTICAL EDITION
January 15, 1942
43
hydroxylamine hydrochloride in the controls by the original amount added. The weights of t h e nickel precipitates of the unknowns are divided b y this factor to ascertain the actual recovery and likewise converted t o t h e equivalent weight of hydroxylamine hydrochloride. These differences from the original amounts indicate the amounts of amine t h a t reacted with the keto groups and when multiplied b y 0.4029 equal the weights of these groups. These weights divided b y the sample weights give the per cent value which is converted to per cent of triketocholanic acid when multiplied b y 4.79.
bonyl groups in bile preparations, the gravimetric procedure based on the recovery of excess hydroxylamine was found to be the more consistent. However, when pigments were absent the titrimetric procedure was preferred because of its timesaving advantages.
Discussion
Literature Cited
The titrimetric method investigated, using a n indicator, has limited use in determining carbonyl groups in the presence of bile pigments. At times the formation of a precipitate obscures the end point. With the gravimetric method the use of a recovery factor t o account for incomplete reactions and losses due t o solubility was necessary. These methods may also be applicable to the various keto steroids related to
(1) Berman, A. L.,Snapp, E., Ivy, A. C., Atkinson, A. J., and Hough, V. S.,Am. J. Digestive Diseases, 7 , 333 (1940). (2) Berman, A. L.,Snapp, E., Ivy, A. C . , Hough, V. S., and Atkinson, A. J., Am. J. Physiol., 131, 752 (1941). (3) Bryant, W. M. D., and Smith, D. M., J. Am. Chem. SOC.,57, 5i (1935). (4) Hirschel, W. N., and Verhoeff, J. A., Chem. Weekblad, 20, 319 (1923).
the hormone field. I n accordance with existing data in the literature (8) it was found that of the natural sources studied, pig bile contains the largest number of carbonyl groups (Table
1). Of the two methods studied for the determination of car-
Volumetric Determination of Bismuth as Caffeine Tetraiodobismuthate (111) ROBERT S. BEALE AND G. C. CHANDLEE School of Chemistry and Physics, The Pennsylvania State College, State College, Penna.
A
XUMBER of organic bases are known to form insoluble
iodometallic compounds from which the metal may be determined b y titration of the iodine from the complex ion. Several determinations of bismuth have been made b y this method, Hexamethyldiiododiaminoisopropyl alcohol ( I ) , naphthoquinoline ( 2 , 8),&hydroxyquinoline (2, 4,10, 12), 8nitroquinoline (S), quinine ( 5 ) , quinoline (6), quinaldine ( 7 ) , dithiocyanato diethylenediamine cobaltithiocyanate ( I S ) , triethylenediamine cobaltichloride (14), and antipyrine methyleneamine (16) form complex compounds with bismuth and iodine and methods using these bases have been reported for the determination of bismuth. Preliminary tests with caffeine showed t h a t i t may be satisfactoxily used t o determine bismuth. An insoluble compound of caffeine, bismuth, and iodine is formed containing the atoms of bismuth and iodine in the ratio of 1 to 4. It was also found t h a t mercury formed no insoluble compound and hence i t was possible t o devise a method for the determination of bismuth in the presence of mercury. So far as i t has been possible to determine, similar methods for bismuth previously reported have all required the absence of mercury.
Solutions C A F F ~ I NSULFATE E SOLUTION.Dissolve 133 grams of caffeine in 570 mI. of 6 N sulfuric acid and dilute to 1 liter; 30 ml. of this solution contain 4 grams of caffeine. CAFFEINESULFATE WASHSOLUTION.Dissolve 1.5 grams of caffeine in 6 ml. of 6 N sulfuric acid and dilute to about 900 ml. Then add 1 gram of pure potassium iodide and bring the volume to 1liter. CAFFEINE KITRATE SOLUTION (for use in the presence of barium ion). Dissolve 133 grams of caffeine in 540 ml. of 6 11’ nitric acid and dilute to 1 liter; 30 ml. of this solution contain 4 grams of caffeine. BISMUTHXITR.4TE SOLUTION. Dissolve 2.79 granls O$ C. P. bismuth nitrate pentahydrate 111 10 ml. of 16 S nitric Had and dilute t o 1 liter. This \\-as standardized with concordant results by the phosphate and carbonate methods (25 ml. of this solution contain approximately 0.03 gram of bismuth). Dissolve exactly 2.0480 grams POTASSIUM IODATE SOLUTIOK. of c. P. potassium iodate t o make 1 liter of solution (1.00 ml. of this solution is equivalent to 0.0010 gram of bismuth). This solution was stable in accordance with studies by Jamieson ( 9 ) .
POTASSIUM CYANIDE SOLUTION.Dissolve 32.5 grams of c. P. potassium cyanide to make 1 liter of a 0.5 M solution. SODIUM HYDROXIDE SOLUTION.Dissolve 5 per cent by weight. POTASSILW IODIDE SOLUTION.Dissolve 8.0 grams of U. S. P. potassium iodide to make 100 ml. of solution. HYDROCHLORIC ACID, 12 N . DIBUTYL ETHER. This must be free from peroxides, so as not to oxidize the iodine in the precipitate.
Procedure Dissolve the sample, containing about 0.03 gram of bismuth, in 10 ml. of concentrated sulfuric acid, remove most of any excess acid by evaporation, and dilute with water to 150 ml. in a 400ml. beaker. (Determinations were made in solutions containing as much as 6.5 per cent acid by volume but 1.5 to 2 per cent is preferable. Since sodium ion does not interfere, the acidity may be regulated, if necessary, by the addition of sodium hydroxide.) Add 30 ml. of caffeine sulfate solution and precipitate the bismuth by the dropwise addition, with vigorous stirring, of 25 ml. of the potassium iodide solution. Let stand for 10 minutes, then iilter by suction through asbestos in a Gooch crucible. It is not necessary to remove the precipitate which adheres to the beaker, since the same beaker is to be used for subsequent o erations. Wash five times with 15-ml. portions of caffeine sulkte wash solution and then four times xith 15-ml. portions of dibutyl ether. Place the Gooch crucible containing the washed precipitate in the beaker in which the precipitation was made, add 100 ml. of the sodium hydroxide solution, and heat to boiling to decompose the precipitate. Cool and add 23 ml. of 12 N hydrochloric acid, then add 8 ml. of the potassium cyanide solution, and titrate with potassium iodate solution to the iodine cyanide end point according to Lang’s method (11). This involves addition of potassium iodate until the color of the solution changes from the brown first obtained to a faint yellow, then addition of 10 ml. of starch indicator, and titration just to the disappearance of the blue starchiodide color. Six determinations on solutions containing 0.0300 gram of bismuth gave 0.0300, 0.0302, 0.0300, 0.0299, 0.0299, and 0.0300 gram by the above procedure. For verj7 small amounts of bismuth, proportionate amounts of reagents were used. Eight determinations on 0.00117 gram of bismuth gave 0.00116, 0.00118, 0.00116, 0.00120. 0.00118, 0,00119, 0.00120, and 0.00119 gram.
