Table I.
Determination of Rhenium with a-Pyridildioxime
Rhenium Rhenium Rel. Comtaken, found, error, rg. 9% pound KReO4 37.2 37.2 0.0 KReOl 74.4 74.2 -0.3 KReO4 112 112 0.0 KReO4" 112 113 +0.9 KReO4 149 150 +0.7 K2ReC16 171 171 0.0 KzReBrs 179 179 0,0 KRe04 186 186 0.0 a 100 mg. of Mo added as XaaMo04. Perrhenate separated by extraction from 531' NaOH into pyridine.
Effect of Reagent Concentrations. T h e maximum color intensity and the time required t o reach it were studied as a function of t h e acid concentration, and the optimum concentration was found t o be 1.2M in hydrochloric acid. At lower and higher concentrations the color intensity was lower and the time required to reach t h e maximum was longer. Color intensity increased with increasing ligand concentration until a ligand to metal ratio of 7 : l was reached.
Beyond this point the ligand concentration had no effect on color intensity. Experiments in which all concentrations except that of the tin(II) chloride were held constant indicated that the optimum concentration of this reagent is 1 gram per 50 ml. of final iolution. Cse of more or less of this reagent lowers the absorbance by a significant amount. Order of Addition of Reagents. T h e order of addition of reagents was unimportant when the rhenium was in the form of the perrhenate ion. K i t h the hexachloro- and heuabromorhenate(IV) ions, more reproducible results \+ere obtained when the ligand was added before the tin(I1) chloride. Effect of Foreign Ions. To determine the effect of foreign ions, 112 p g . of rhenium as the perrhenate were treated with 100 mg. of the foreign ion and the recommended procedure b a s followed. Those ions causing less than 2y0 relative error were Pb(II), Al(Ill), Ri(III), K(I), Mg(II), Cd(II), Mn(II), Zn(II), Sr(II), and acetate. Those ions causing more than 27, but less than 5% relative error were Cu(II), Sb(III), sulfate, phosphate, and citrate. X relative error of less than 557, resulted from Ni(I1) if twice the
recommended amount of ligand was used. I relative error of leqt than 2% resulted with Cu(I1) under the .ame conditions. While 100 mg. of Fe(II1) and Co(II) caused relative error5 of lo%, 50 mg. of Fe(II1) or 25 mg. of Co(I1) caused relative errors of less than 27,. Complete interference resulted from the presence of Cr(III), molybdate, oxalate, and thiocyanate. LITERATURE CITED
( 1 ) ;Ileloche, V. l f . , llartin, R. I,., Webb, W. H., .%NAI,. CHEM 29, 52i
( 1957). (2)-Meshri, D. T., Haldar, R . C., J . S e i . Ind. Res. Indza 20B, 551 (1961). ( 3 ) Trask, W. T., Jr., Ph.D. thesis, Iowa State University, '1957. (4) Tougarinoff, M. B., Rvll. Soc. Chim. Relges 43, 111 (1934). ( 5 ) Tribalat, S.,Compt. Rend. 224, 469 (1947).
FREDTRCSELL~ RICHARD J. THOMPSOX
Department of Chemistry Texas Technological College Lubbock, Texas WORK carried out under Grants D-094 and D-097 from The Robert A. Welch Foundation, Houston, Texas. Present address, Marathon Oil Co., Denver Research Center, Littleton, Colo.
Spectrophotometric Determination of Glycyrrhizic Acid in Licorice Extract Sir: I n the L-nited States the tobacco industry is the largest user of licorice extract, Succus liphii!iae, which serves as a characteristic :flavorant and a moisture-controlling agent in tobacco casing solutions. The active principle of licorice is a sweet-tasting material rrhizin, a glycoside in the otassium ;and calcium salt form of glycyrrhizic acid. Proper evaluation of licorice estract is dependent in part on accurate determination of the glycyrrhizic acid content. Xieman ( 3 ) , in his excellent study, gives not only the means of assay and chemical structure for glycyrrhizin, but other details on the licorice root and estract including its pharmacology. Thp most widely used technique for rrhizin determination is the acid precipitation proce Houseman ( 2 ) . T h painstaking, and larks both accuracy and specificity. The most promising; method for specific determination of glycyrrhizic acid is to hydrolyze it to its aglycon, glycyrrhetinic acid, and after extraction determine the aglycon eit,her by colorimetric, ultraviolet, or polarographic means. W e a t (5) and Brieskorn and blahran ( 1 ) describe colorimet,ric meth-
ods, whereas Onrust, Jansen, and Wostmann (4) describe a polarographic method. These methods were investigated a t considerable length, and the procedure finally evolved was a modification of the colorimetric procedure described by Wiest (6),using a modification of Onrust, Jansen, and Wostmann (4) to hydrolyze glycyrrhizic acid to glycyrrhetinic acid. EXPERIMENTAL
Reagents. Vanillin, 1% in absolute ethanol. Store in an amber bottle. Glycyrrhetinic acid, st,andard. No commercially available material was of sufficient purity for use as a standard. Prepare by dissolving 5 grams of ammonium glycyrrhizinate in 500 ml. of 50y0 dioxane (v./v.) and reflux with 100 ml. of 5 N sulfuric acid for 3 hours. Extract with chloroform and evaporate the chloroform solution to dryness under reduced pressure. Dissolve the residue in ethanol, char-treat, and filter. Heat the filtrate to boiling point, add water until slightly turbid, remove from heat, and allow to crystallize. Recrystallize two additional times in similar manner and drv at 110" C. Melting point (uncorr.), "294-297' C. Purity by nonaqueous titration, 100.28'%.
M o n o a m m o n i u m glycyrrhizinate MacXndrews and Forbes Co. Purified by two recrystallizations from dilute ethanol. Purity by nonaqueous titration, 99.82%. Hydrolysis Procedure. A4ccurately weigh 0.1 gram of licorice into a 250ml. T Erlenmeyer flask, add 20 ml. of 50% dioxane, and connect to a reflux condenser. Apply heat and when the licorice is dissolved, add 20 ml. of 12N sulfuric acid through the top of the condenser. Reflux vigorously for 1 hour, add 70 ml. of water, then 100 ml. of chloroform t,hrough the top of the condenser. Reflux an additional 15 minutes, cool, then transfer the contents of the flask t'o a 250-ml. separatory funnel. Shake vigorously, allow the phases to separate, and draw the chloroform layer into a second funnel containing 100 ml. of 2% sodium bicarbonate. Shake vigorously, and after phase separation drain the chloroform layer into a chromatographic tube containing 25 grams of sodium sulfate, collecting the filtrate in a 200-ml. volumetric flask. Repeat the extractions with 75, then 25 ml. of chloroform, finally adjusting the volume of the filtrate to 200 ml. Analysis for Glycyrrhetinic Acid, Pipet 4.0 ml. of the chloroform dilution into a 20- X 200-mm. test tube, and 4.0 ml. of chloroform into a second tube to serve as a blank. VOL. 36, NO. 9, AUGUST 1964
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ucts, the chloroform solution has a yellow color which varies in intensity with the licorice being analyzed. This foreign color definitely interfered in color development, giving a brownred color as opposed to the desired red color. As a result, high, imprecise results nere obtained. This yellow color could be removed by char-treatment, however, varying amounts of glycyrrhetinic acid were also removed by all char-treatments tried. Most of the hindering yellow color was removed by washing the chloroform extract with 2% sodium bicarbonate as described above. I n this manner none of the glycyrrhetinic acid was lost, and the spectra from analysis of licorice samples were comparable to that from pure glycyrrhetinic acid. A single extraction with bicarbonate was sufficient to remove the interferants, as identical results were obtained after additional bicarbonate extractions Analysis of Ammonium Glycyrrhizinate and Recovery of Ammonium Glycyrrhizinate after Addition to Licorice. This procedure was evaluated and proved by analyiis of known amounts of pure ammonium gljcj-rrhizinate, and by adding known amounts of the compound to licorice samples, analyzing, and correcting the results for the amount of glycyrrhizic acid present in the licorice. Table I demonstrates that quantitative recovery of ammonium glycyrrhizinate wa. obtained, both from pure solution
