(12R) King, R. W.,Hirschler, -1. E., Ibid., 26, 1397 (1954). (13R) Knight, H. S., Groennings, S., Ibid., 26, 1549 (1954). (14R) Alair, B. J., Pignoco, .4.J., Rossini, F. D., Ibid., 27, 190 (1955). (15R) Mrlpolder, F. W.]Washall, T. A., Alexander, J. A., Ihid., 27, 974 (1955). (16R) lfende, H., Ghnie chim. 75, 1 (January 1956). ( 1 i R ) llilner, 0. I., Liederman, D., h S A L . CHEM. 27,1822 (1955). (18R) Mosely, J. R., Lucchesi, C. A., Mueller, R. H., Ibid., 27, 1440 (1955).
REVIEW OF INDUSTRIAL APPLlCATlONS
(l9R) Serheim, -4.G., Dinerstein, R. A , , Ibid., 28, 1029 (1956). (20R) Kester, R. G., Ibid., 28, 278 (1966). (21R) Sewchurch, E. J., -1nderson, J. S., Spencer, E. H., Ibid., 28, 154 (1956). (22R) Perry, E. S., Cox, D. P., I n d . E n g . Chern. 48, 1473 (1956). (23R) Post, B. G., Baker, M. O., Hiett, T. A , , Murphy, J. L., ANAL. CHEM.26,617 (1954). (24R) Rasmuseen, D. J., O ~ C .Dig. Federation Paint & Varnish Prodirction Clubs 27, 529 (1955).
(25R) Rot’hard, D., Petroleum Processing 10,
1914 (1955).
(26R) Schaefermeyer, W.C., Smith, E. S., rlN.4L. CHE3r. 27, 1040 (1955). (27R) Shoolery, J. S . , Ibid., 26, 1400
(1954). (28R) Sullivan, L. J., Ruppel, T. C., Killingham, C. B., I n d . Eng. Chem. 47, 208 (1955). (29R) Tunnicliff, 11. l l , , Stone, H., .$lsa~. CHE3f. 27, 3:; (1955). (3OR) P a n Schooten, J., Van Xes, K., Rec. hac. chim. 73, 980 (1951). (31R) Winters, J. C., Dinerstein, R . A., .kX.IL. CHEM. 2 7 , 5 4 6 (1955).
I I I
I Pharmaceuticals and Related Drugs I I I
I
R. P. HAYCOCK and W. J. MADER Research Department, Ciba Pharmaceufical Producfs lnc., Summif, N. J.
T
R W I E W covers the analytical piocedures reported in the readily available journals foi 1955 to July 1956, and in addition. articles abstracted by HI\
Chemical Abstracts. Analytical Abstracts. 01 the Zeitschrijt f u r analytische Chenaie for this period. However, a
consideiable selectivity has been exercised and this review does not attempt to cover all of the many contributions. I n some instances the authois have used the anal! tical procedures reported in this ieviei~; in others. the comments are neccsssiily limited to theoietical discussions. Gieatei emphasis is placed on specific, accurate, and reliable analytical procedures for pharmaceutical analysis; piocedures nhich can be correlated to the biological activity of the drug. The proof of the stability of a pharmacential product is of utmost importance and the majority of the papers published ieflect this attitude on the part of pharmaceutical analysts. ALKALOIDS
Morphine and its derivatives and strychnine have been analyzed by paper chromatographic procedures (72, 451 , 467). i\lixt,ures of butanol-glacial acetic acid-water and butanol-formic acid are used as solvents; morphine is developed with ferric chloride-ferricyanide reagent, and strychnine with a modified Dragendorff reagent. A method of planimetric evaluation of the A developed spots was described. colorimetric method for the deterniina-
tion of morphine is based on reaction with nickel chloride (496). The reagent is added to the alkaloid solution after treatment with iodic acid (HIOB) and the addition of ammonium acid carbonate-ammonium chloride-ammonium hydroxide buffer. The absorption of the solution is measured a t 670 and 530 mp. The British Pharmacopoeia and G. S. Pharmacopoeia procedures for morphine in opium were criticized for the lack of complete extraction and simultaneous extraction of other alkaloids (532). The author claims that the method of Eder and Waeckerlin is satisfactory. Morphine sulfate can be separated from atropine sulfate by ion exchange chromatography using a t\vo-bed column of Amberlite I R 4 B and rlmberlite IRL4-410 (50). A method for isolation and purification of morphine froni poppy was reported (3). Yoshino and Sugihara (559) separated nicotine, strychnine, and brucine by ion exchange chromatography. The alkaloids are adsorbed by a m-eakly acidic exchange resin and then eluted fractionally with 0.1 to 1 M ammonium chloride. The separation of strychnine and brucine \vas incomplete. An oscillographic polarography technique for the identification of opium alkaloids has been described (156). A table of voltages at which the depressions occur in the polarography and the oscillographic polarograms for some of the compounds and mixtures are given. A niicrochemical differentiation of morphine and nalorphine (367) has been
outlined, using Marme’s cadmium iodide reagent, Kagner’s iodine reagent, pure hydriodic acid (HI), and picrolonic acid. The alkaloids niay:be separated by paper chromatography; RI values and diffraction data are also given. Micro quantities of strychnine are determined by a n ainperonletric method (346). The procedure is based on the observation that the height of the polarographic reduction wax-e of tungstosilicic acid is linearly reduced by the addition of strychnine. Strychnine, atropine, cocaine, and some other alkaloids we chromatographed as free bases by using as solvents aqueous solutions of ammonia, methylamine, and ethylamine (84). The R f values obtained with various concentrations of the amines named, using ascending and revolving-disk methods, are tabulated. The principal alkaloids of opium have been analyzed by a spectrophotometric method (136). Van Etten and associates (624, 625) determined morphine in Papaver somnijerum by adsorption on ion exchange resins and estimation by conventional procedures, such as the nitroso colorimetric method. ultrariolet absorption, and titration. Polarometric titrations of alkaloids with picric, picrolonic, styphnic, and flavianic acid and sodium alizarinsulfonate v-ere reported (566). Kone of the acids indicated gave satisfactory results with all alkaloids tested. A colorimetric method for the determination of papaverine is based on condensation with formaldehyde (47.2). The condensation 1 roduct is treated VOL. 29, NO. 4, APRIL 1957
697
with bromine water, ethyl alcohol, and then with aqueous ammonia solution. The blue-violet color is specific and is measured colorimetrically. Polarographic determination of papaverine, using tetramethylammonium hydroxide, is suitable for determination in mixtures with codeine ( 4 4 . Ultraviolet techniques for determination of papaverine. morphine, and ephedrine (504, and for the determination of mixtures of papaverine and codeine (505) have been described; a n infrared method for the estimation of papaverine was also reported (430). %. conductimetric titration of certain alkaloids with naphthalene-2-sulfonic acid in an acetonic solution was reported by Udovenko and Uvedenskaya (517). The method depends on the effect of acetone in increasing the differences between the ionization constants of the bases. A fluorometric technique for the determination of ergot alkaloids has been found (192, 193). -4 blue fluorescence is developed by treating the alkaloids with ethanolic ethane-sulfonic acid. Hydrogenated ergot alkaloids do not show fluorescence. The spectrophotometric differentiation of ergot alkaloids from luniilysergic acid has been described (379). Salts of ergot alkaloids can be titrated with perchloric acid in anhydrous acetic acid (191). To determine the acid component, the sample was dissolved in pyridine and the solution was titrated with potassium niethoxide. Systems for the paper chromatographic separation (297, 300, 380) and the microscopic examination of ergot alkaloids were reported (267). A spectrophotometric determination of heroin and quinine is based on the ultraviolet absorption a t 297.5 and 330 mp (385). At the former wave length, equimolar concentrations of the two alkaloids have almost the same absorption, while a t 330 nip the absorption is due entirely to quinine. Adrenaline and noradrenaline may be separated by paper chromatography (81). Using a circular horizontal technique and methanolic-aqueous tartaric acid solvent, R, values of 0.75 and 0.68, respectively, were obtained. Noradrenaline develops a green fluorescence when condensed with ethylenediamine (148). Stock and Hinson (481) made spectrophotometric studies of the iodate and persulfate oxidation of adrenaline and noradrenaline. Their data may be utilized for a n approximate assay. Another procedure for the analysis of solutions of epinephrine and norepinephrine (542) depends on conversion of the substances into the triacetyl derivatives which are extracted into chloroform and examined polarimetrically. Results show good agreement with U. S. Pharmacopoeia dog bioassay. B y supplementing the method with partition chromatography of the acetyla698 *
ANALYTICAL CHEMISTRY
tion product, the norepinephrine may be isolated as its triacetyl derivative and determined colorimetrically after diacetylation and oxidation to noradrenochrome. Mixtures of nicotine, nornicotine, and total alkaloids have been analyzed by extraction of an alkaline tobacco mixture with benzene-chloroform and titration with perchloric acid-anhydrous acetic acid (111). The fact that secondary base-type alkaloids may be quantitatively acetylated provided a means of determining nornicotine-type alkaloids in the presence of nicotine. Three chromatographic methods for determining nicotine and alkaloids in tobacco nere reported (231,984). An improved separation is achieved by pretreatment of the paper with p H 5.6 acetate buffer. A turbidimetric determination of nicotine, based on T’alser’s reagent, is in close agreement with the Association of Official Agricultural Chemists’ (AOAC) procedure (401). The nicotine is precipitated with potassium mercuric iodide and measuied turbidimetrically by coniparison with standards; nornicotine does not interfere. Scott and associates (455) have reported a convenient physical method for the determination of hyoscine in Benadryl-hyoscine tablets. The tablets are extracted with ethyl alcohol, evaporated to dryness, and taken u p in dimethylacetamide for measurement of the infrared absorption. Benadryl hydrochloride does not interfere because of limited solubility. A colorimetric procedure for atropine based on a modification of the VitaliRIorin reaction was investigated (169). The alkaloid \vas nitrated with fuming nitric acid, dissolved in dimethylformamide, and treated with tetraethylammonium hydroxide. The absorption of the solution was determined a t 540 mp. The method is not specific and is also applicable to phenylacetic acid, benzylpenicillin, benzathine penicillin, and chloramphenicol. Rosenblum and Taylor (416) have criticized the U. S. Pharmacopoeia and British Pharmacopoeia identity tests for apoatropine and belladonnine in atropine-on the grounds that the tests are sensitive for belladonnine but allow the presence of appreciable amounts of apoatropine. The authors propose a potassium permanganate test which is sensitive for apoatropine but does not react with belladonnine. Reichelt (391) impregnated paper with formamide and a solvent of chloroform or benzenechloroform to separate the belladonna alkaloids. The alkaloids are eluted from the paper with ethyl alcohol, acetic acid, and water and determined colorimetrically by the Vilde reaction. Mass analysis technique has been applied (378) to the analysis of strychnine, brucine, atropine, and hyoscyamine.
A scheme to identify atropine or scopolamine as well as the opium alkaloids in injectables has been reported by Sakurai and Chiba (498). Chromatographic techniques for protoveratrine were reported (184, 286). I n the former technique, protoveratrine A and B are separated by paper partition chromatography, eluted, and determined colorimetrically by a conventional method. The latter procedure is based on passage of the sample in chloroform over a Celite column containing buffered chlorophenol red as the immobile phase. A quantity of dye equivalent to the concentration of the alkaloid is removed. A reflectometric method for nieasuring density values of paper chromatograms of alkaloids was proposed by Thies and Reuther (503). The bismuth sodium iodide reagent, which gives a red color with alkaloids, was used. The same authors (509) improved the solvent mixtures for the separation of alkaloids by paper chromatography by the addition of an ester. They note that esterification of solvent mixtures can be largely prevented by adding the corresponding ester to the solvent mixture. The National Formulary procedure for Xux Vomica tincture has been shortened (454). Preliminary isolation of strychnine and brucine is by adsorption on aluminum oxide (A1203), followed by elution with i O % alcohol. The eluate is subjected to ion exchange and subsequent elution with 0.1 N hydrochloric acid in alcohol. Strychnine and brucine are determined a t 255 and 264 mp, respectively. Head and associates (907) have demonstrated that ultrasonic extraction and maceration of cinchona succirubra followed by Soxhlet extraction are more efficient than Soxhlet extraction alone. A new procedure was described for the assay of cinchona which is based on the precipitation of the majority of the nonalkaloidal emulsifying substances by the addition of calcium peroxide (CaOz) (307). A note on the determination of codeine in aspirin, phenacetin, caffeine, and codeine tablets recommends a modification: The codeine is extracted with chloroform from a basic solution and titrated with perchloric acid in dioxane to a colorless end point using chlorophenol red indicator (370). Caffeine can be titrated with 0.lN perchloric acid-acetic acid ( 7 ) . A recent approach to the assay of alkaloidal crude drugs was described by Brochmann-Hanssen (68, 69). The alkaloids are extracted and separated by means of ion exchange resins and determined by spectrophotometric procedures. A microchemical identification technique for alkaloids was reported (99). The crystals obtained from 30 different alkaloids, using various reagents, were tabulated. Modi-
fications of several color tests were also using p H 3.5 buffer; the location of the described. Analytical procedures have alkaloids was detected with bromobeen reported for sanguinarine (437). phenol blue dye (685). Methods have sinonienine (S16), galegine (249), and been described for the chromatographic lupine (336). separation on paper of iodinated tyroThe RauwoZJia alkaloids have resines and thyronines by using as solceived much attention since the isolavents aqueous methanol or ethyl alcohol tion of reserpine was reported in 1952. buffered to p H 7.4, 7.8, or 6.2 with Chromatographic techniques, both on or ammonium carbonate [(SH.t)zC03] paper and column, have been used exammonium acetate (402). The partition tensively. Paper chromatographic coefficients between 0.2-If phosphate procedures have been described for buffer of different p H values and chlorochecking the purity of commercial samform for different alkaloidal constituents ples of RauwoZJia alkaloids and the assay of R. serpentina have been described of mixtures used for therapeutic pur(21). The microscopic differentiation poses (61). RI values are given for of roots of R. serpentina from other ajmaline, deserpidine, serpentine, yospecies has been reported by Eisenberg himbine, ajmalicine, and corynanthine. and Schulze (142). An Indian journal Reserpine has been separated in phar(374) discusses variations in the alkaniaceutical preparations by chroniatoloidal content of Rauzroljia fioni difgraphic adsorption on Solka-floc and ferent sources and describes an ?.;trapCelite (1 to l),eluted with 5 S acetic acid, tion-giavimetric technique for alkaand determined by ultraviolet ahsorploidal content. The authois found the tion (31). A method was described for British Pharmaceutical Code.; assay the estimation of reserpine based on the satisfactory for routine control only. spectrophotometric determination of A fluoronietric method for the deter3,4,5-trimethoxybenzoic acid liberated mination of reserpine in tablets and exby saponification (128). The triniethtracts of Rauzroljia serpentina is based oxybenzoic acid was chromatographed on the reaction with hydrogen perouide on paper with a solvent consisting of (120). The intensity of fluorescence equal volumes of 2% aqueous ammonia is proportional t o the Concentration , and butanol and eluted with 0 . W amof reserpine. The author clainis that monium hydroxide. The ultraviolet abthe reaction is specific. RIannell and sorption was measured a t 253-4 and 234 Allmark (306) made a comparative nip. Banes, Carol. and Kolff (24) have study of two methods of assay for redescribed a chromatoglaphic method for serpine in commercial tablets. The the annlys;s of commercial reserpine method based on ultraviolet absorption preparatio1,s which separates reserpine was found unsuitable for some brands of from closely related compounds and perieserpine tablets. The second method, mits its assay by spectrophotometr!.. based on fluorescence, v-as found satisThe same authors (83) reported a techfactory for the analysis of reserpine in nique for the chemical identification and tablets, elixirs, and injections. A chemassay of RaziicolJia serpentina root and ical method for the diffeientiation of its preparations. Rauu~olfiaserpentina roots of R a u w o l g a serpentina from the is differentiated from other Rauwolfia roots of other RauwoZjia species (23) species by paper chromatograms. Uriis based on the ultraviolet absorption nary indoles have been detected by a spectrum determined from 250 to paper Chromatographic procedure (834). 370 nip. as compared n-ith the spectra The substances are separated on K h a t of 3,4,5-trimethoxgbenzoic acid and man S o . 1 paper using a niixtuie of 3,4.5-trimethoxycinnamic acid, the acids propyl alcohol and aqueous ammonia by formed by the hydrolysis of reserpine a vertical technique and are chromatoand recanescine. A colorimetric degraphed a t right angles with butanol termination of 5-hydroxyindol-3-ylacetic acid (6HIAA) is based on the pand aqueous glacial acetic acid. The dimethylaminobenzaldehyde reaction indoles a1 e detected Tyith p-diniethyl(104). The reagent in concentrated ainiriohcnzaldcliyde. hydrochloric acid is added to a solution llecher (121) has modified Ehrlich's of 5-hydrouyindol-3-ylacetic acid and aldehyde I eagent (p-dimethylaminobenzheated for 10 to 12 hours a t 56" to 58' C. aldehyde) and has increased its sensiThe absorption is measured a t 600 mp. tivity for the detection of urinary indiThe author also determined 5-hydroxycans. indicanoids, and similar metaindol-3-ylacetic acid by ultraviolet abbolic pioducts of skatole and 2-methylsorption after adjustment to p H 7.0. indole. After the application of EhrThe extinction was measured a t 221 lich's reagmt, the chromatogram is mp. Details of a colorimetric technique splayed n-ith aqueous ammonia and exfor the assay of 5-hydroxy-3-indoleaceamined under ultraviolet light. The tic acid, a metabolite of 5-hydroxyappearance of a fluorescence is termed tryptamine with 1-nitroso-2-naphthol rethe fluorindal reaction, which is more agent have been presented by Udensensitive than the conventional refriend and coworkers (616). agent. Veratrum alkaloids have been Three methods for the determination separated by paper chromatography of reserpine and the physical properties
of reserpine have been discussed (801). The first method is based on ultraviolet absorption a t 268 and 295 mF after removal of reserpic acid and trimethoxybenzoic acid by evtraction with 0.01M hydrochloric acid and sodium acid carbonate solution, respectively. The second method is a colorimetric method based on heating reserpine with strong organic acids; the absorption is measured a t 380 mp. I n the third method, the fluorescence which developed on heating reserpine n i t h a strongly acid solution forms the basis for the determination. d colorimetric technique for the assay of reserpine in pharmaceutical products is based on the formation of a chloroform-soluble complex with bromophenol blue (60). The extinetion is measured a t 402 mp against chloroform as a blank. Quaternary ammonium conipounds, some antihistamines, and other organic bases interfere. I n the same journal, Banes has proposed another colorimetric method for the determination of reserpine using a conventional reagent. vanillin (22). The alkaloid may be hydrolyzed and the resulting reserpic acid and triniethoxybenzoic acid may he determined by means of the vanillin reagent and ultraA violet absorbance, respectively. color reaction for reqerpine haqed on the reaction n i t h p-dimethylamino benzaldehyde in ethyl alcohol containing sulfuric acid was described (114). A report on the crystallography of reserpine and reserpic alcohol hydrate (410) ; ajmalicine, ajmalicinc hydrate, and py-tetrahydroserpentinol (411 ) ; and yohimbine (412) have been reported by Rose, and Bose reports some chemical and physical properties of serpinine (62). The chemistry of ll-desniethoxyreserpine has been described by Harrisson and coworkers (201). The physical properties and structure of raunescine and isorauncqcine TI ere reported (222). Earl, Kinters. and Schneider reported an unusual biotechnique for the determination of Serpasil based on emesis in pigeons (IS?'). l n ultraviolet spectrophotometric procedure for the assay of reserpine has been described ( 6 2 7 ) . A colorimetric procedure for the determination of tryptophan i q based on the characteristic red color produced 7% hen tryptophan is layered on a solution of ceric sulfate [Ce(SO,),] in sulfuric acid (8); another quantitative colorimetric technique for tryptophan is baqed on Eckert's reaction (477). AMINO ACIDS, AMINES, AND RELATED COMPOUNDS
Paper chromatographic techniques for the separation of amino acids have received added emphasis. Underwood and Rockland (520) obtained the most satisfactory separations with tert-butyl VOL. 2 9 , NO. 4 , APRIL 1957
699
alcohol-water-formic acid and phenol -water-ammonia. A technique using ninhydrin-copper complex as the color reagent 11-as reported (67) and another using conventional ninhydrin reagent was described (568). The quantitative determination of amino acids by circular paper chromatography has been used (272). These authors separated 16 amino acids on a series of chromatograms by using different solvent mixtures and ninhydrin as a color reagent. Rf values of amino acids, using different one-phase solvent mixtures, have been determined (200). Oreskes and Saifer (352) employed a circular chromatographic technique using phenol and butanol-acetic acid as solvents with isatin and ninhydrin as color reagents for the determination of amino acids in protein hydrolyzates. The chromatography of a-keto acids and their hydrogenation products x i s reported (317 ) . Clayton (101) has investigated the relationship between chamber size and Rf yalues of amino acids. The author uses a new term, “critical volume,” which defines the volume of solvent necessary to saturate a specific chamber. The most reproducible chromatograms are obtained when small chambers are used because the amount of solvent generally exceeds the critical volume of the chamber. An extraction procedure for amino compounds in biological materials involves the combined use of adsorption and dialysis (226). Kalant applied a photometric ninhydrin reaction to the measurement of amino nitrogen in plasma (246). Neutral equivalents of amino acids and peptides can be determined in alcohol or aqueous alcohol by titration with alcoholic potassium hydroxide and proper indicators (149). Two-dimensional paper electrophoresis has been employed in the separation of amino acids (316). The infrared spectra of 3-phenyl-2-thiohydantoins of amino acids and their application to the identification of IT-terminal groups in peptides was reported (387). d colorimetric method for the determination of arginine is based on the reaction with sodium hypobromide, 1-naphthol, and urethane (268). The oscillopolarographic detection of amino acids has been described by Pechan and coworkers (366). If care is taken, the method may also be applied quantitatively to very small amounts of substance. A color method for the detection and determination of amino acids has been developed; 1,2-diacetylbenzene, 1,2dipropionylbenzene, and l-acetyl-2propionylbenzene react under appropriate conditions mith amino acids, producing dyes (398). A modified ultraviolet spectrophotometric microtechnique for the determination of amino acids and peptides as copper complexes has been described by 7M)
ANALYTICAL CHEMISTRY
Cherkin and associates (92). Amino acids have been separated by circular paper chromatography and identified by spot testing on paper using conventional reagents-Le., alloxan, isatin, and ninhydrin (425). Schweet has described a colorimetric procedure based on reaction with hydroxamic acid and ferric chloride (452). The adsorption is measured a t 520 mp after 5 to 20 minutes. Amino acids, after oxidation with periodate, are detected on chrom:btograms with benzidine and starch-iodide reagent (97). An electrophotometric method for determination of amino acids by paper chromatography is based on the measurement of the size of the spots and their color intensity (96). The value obtained from the determination of one amino acid can be used for the determination of any other amino acid, by simply multiplying by a molecular weight factor. Amino acids have been separated and determined by “test tube paper chromatography” (282). Mixtures of phenol and water and ternary mixtures of tert-butyl alcoholmethanol-water are used as solvents. Test tube chromatography can be used a t higher temperatures and gives better and faster separating effects. Good results are claimed for paper chromato- ‘ graphic determination of glycine, tryptophan, and glutamic acid in medicaments using phenoI n i t h aqueous ammonia as solvent, ninhydrin in butanol as developer, and heating a t 80” to 90” for 5 minutes (529). A specific spectrophotometric method is used for the determination of proline in protein hydrolyzates, urine, and plasma. Interfering basic amino acids are removed by shaking with Permutit; hydroxyproline does not give the color reaction (510). Ninhydrin is added to an acetic acid solution of proline and heated, and the extinction of the benzene extract of the color complex is measured a t 515 mp. The principal factors influencing the colorimetric determination of hydroxyproline by reaction with p-dimethylaminobenzaldehyde were studied by Meyada and Tappel (320). A convenient assay method has been developed which depends on the combination of histamine and histidine with carbon disulfide and the polarographic behavior of the reaction products (662). By paper ionophoresis, histamine and histidine are separated with a barbital buffer solution a t pH 8.6. The eluate of each deposit was determined polarimetrically. Histamine has been isolated by paper chromatography using a solvent mixture of butanol-acetic acid-water or isobutyl alcohol saturated with aqueous ammonia (449). The paper was then dried and sprayed with p H 11 buffer and diazotized sulfanilic acid. The reddish spots of histidine and histamine can be eluted and
quantitatively evaluated. A method for the determination of micro amounts of histamine has been rendered more sensitive by coupling with diazotized sulfanilic acid a t pH 9.8 and a t O’C., ethyl alcohol being added to stabilize the color (442). A sensitive paper reaction of ninhydrin with hydroxyproline and proline was outlined (100). A polarographic determination of histidine and di-dinitrophenyl histidine mas reported (543). Another paper chromatographic procedure for histamine, using butanol-acetic acid and diazotized sulfanilic acid, was described (166). Schayer and associates reported on the use of radioactive iodine for the determination of histamine (436). Paper electrophoresis has been employed in the separation and determination of amino acids (446). Identification and measurement of the compounds were accomplished by staining with ninhydrin and taking photometric measurement of the spots. Another ninhydrin procedure was described for the estimation of glutamic acid on buffered paper (260). A modification of the Knoop reaction forms the basis for the analysis of histidine (226). The method takes into account the effects caused by the concentration of bromine during bromination and the temperature a t which the color is developed. A micromethod for the determination of tryptophan is based on the fluorescent intensity of substances formed by reaction with glucose a t a controlled p H and temperature (344). Tryptophan is separated from other amino acids on a resin column of Dowex-50. Conditions n-hich enhance the solubility of cystine and tyrosine in chromatographic solvents and result in adequate separation were outlined (484). The determination of amino acids in plant proteins using Sanger’s l-fluoro-2,4-dinitrobenzene method was reported (126). The amino acids thus obtained were identified by paper chromatography in citrate buffer and in 2-butanol-butyl acetate-hydrochloric acid. Hippuric acid, a-amino, Pamino, and -V-acetylamino hippuric acid have been separated by paper chromatography and determined colorimetrically with p-dimethylamino-benzaldehyde in acetic anhydride (195). Ornithin has been isolated and determined from gelatin hydrolyzate by chromatographing on Dowex-50 (198). A specific and sensitive method for the determination of microgram quantities of uracil and thymine (171) depends on treatment of pyrimidines with labelled p-iodobenzenesulfonyl chloride in acetone, a t room temperature, buffered a t pH 8.5 to 9.0 with tetramethylammonium bicarbonate. A colorimetric procedure for the determination of 2-hydroxy-4, 6-dimethylpyrimidine is based on reaction with sul-
fanilic acid and sodium nitrite in a n alcoholic sulfuric acid solution (489). The absorbance is measured at 540 inp. A specific test for antipyrine and 1-naphthylamine is based on condensation of nitrosoantipyrine in a n acetic acid solution with 1-naphthylamine hydrochloride (158). Satisfactory re-ults n r r e obtained in the assay of antipyrine and its salts (314). The compound n a s treated with nitrite in acid solution to obtain 4-nitroantipyrine. n hich \vas submitted to polarography. Sitrofurorone in feeds has heen deterniincd by treatment of a dioxane solution with alcoholic sodium hydroxide (506). Szalkowski and Mader have re\ iened the AOAC procedure for the clcterniination of sulfaquinoxaline in mcdicated feeds (491). The uqe of ficin, a proteolytic enzyme. relea-e. the bound sulfaquinoxaline and enables a iiiore quuntitative deterniination of the drug. Sicarbazin (44’dinitrocarb:inilide - 2 - hydroxy - 4,B - dimethylpyrimidine) is extracted from the feeds by c~iniethylformamide-the e\tr:ict it chromatographed and the Sicarhazin i:. detcmnined colorimetrically (-$sa),tht, 2 - hydroxy- 4,B - dimethylpyriniidint. liy reaction with sulfanilic acid arid sodiuni nitrite in dilute sulfuric acid. arid the 4,4’-dinitrocarbanilide by a 11 ell-knon 11 technique for determining dinitro compounds. 2- and 3-Chloroxnnthydrol TI ere reported as reagents for :iniid(,s; hou w t r . neither reacted with amides its rusily as did xanthydrol (514). Mononitro compounds produce orange. red, or purple colors in dimethylformide upon the addition of tetraethylammonium hydroxide (384). Sitroguanidine and thiourea can be titrated with perchloric acid in trifluoroacetic acid but not in glacial acetic acid (127). The detection of urea and hydroxybenzaldehyde is based on heating n ith diniethylglyoxime and thiosemicarbazide to produce a red or red-purple color (349). The decomposition of nitrohydroxy compounds into fornialdehyde, lvhich combines n-ith chromotropic acid to form a violet colored complex in sulfuric acid, was used as the l m i s for a colorimetric technique (239). -4procedure for determination of biuret in urea pyrolyzates based on the copper biuret complex has been described (1 $$). A method for analysis of benzidine hy reaction with potassium perniangnnate in nitric acid to form a grcwish yellon- color, which is measured color~inetrically, has been reported (SQT). Siggia and Stahl determined amides by reduction to the amine n ith lithiiiru aluminum hydride. The amine is steam-distilled and titrated with standard acid (467). A coulometric determination of isonicotinic acid hydrazide by electrochemical oxidation nith chlorine n a s used (248). A milli-
ammeter and iodine coulometer are placed in series with the electrolyzed solution and the liberated iodine is titrated n-ith 0.01S sodium thiosulfate. A colorimetric method for isonicotinoyl hydrazide n-as based on reaction Ti-ith sodium p- 1,2-naphthoquinone-4-monosulfonate, sodium carbonate, and hydrazine hydrochloride ( 6 7 ) . The absorption was measured a t 500 mp. Svoboda (487) reported a simple polarographic method for pyrazinamide; iqoniazid does not interfere. Dilute aqueous solutions of ethyleneimine or n-butylamine react m-ith 1,2-naphthaquinone4rsulfonate a t p H 10.3 to give chloroform-extractable reddish dyes (415). ilbsorbance measurements are made a t 420 and 450 mp, respectively. Epstein and con-orkers reported (145) the use of 4-(4nitrobenzyl)-pyridine in the colorimetric determination of rthylenpimines and various alkylating agents. 1 procedure based on reaction betFveen an ethyleneimino group and thiobulfate a t pH 4 has been described ( 5 ) . A colorimetric determination of secondary aminrs has been described (536). I n a n alkaline solution. secondary amines react with sodium nitroprusqide to give a violet-blue compound. Conditions for quantitative determination were described. Critchfield and Johnson (110) deqcribed a colorimetric technique for the determination of primary aliphatic amines. The amine react. u-ith copper chloride, salicylaldehyde, and triethanolamine. The copper-salicylaldehydeimine formed is extracted with hexanol and the amount of copper is determined by reaction n4th bis(2-hydroxyethyl) dithiocarbamic acid. The same authors titrated the dithiocarbamic acid formed by the reaction of amines with carbon diwlfide using phenolphthalein and thymolphthalein indicators (109). d micromethod for determining primary aromatic amines by potentiometric titration with sodium nitrite has been describrd (289). The method is baqed on the use of potassium bromide as a catalyst. A procedure for secondary aliphatic amines liaqed on formation of the nitrosamine and measurement of the absorbance has been reported (98). A color reaction of 1-phenyl-2-dimethylaminopropane and other tertiary amines was reported (547). Acetic anhydride solutions or citric, aconitic, and malonic acids produce a red, violet, or hlue coloration with tertiary amines on heating. Propamine and methylpropamine upon nitration yield nitro compounds suitable for polarographic determination (318). The same technique has been used on antipyrine. A volumetric method for ethylenediamine was based on reaction with formaldehyde (123). A Schiff base is formed with the liberation of acid which is titrated with sodium hydroxide. Acriflavine behaves polaro-
graphically as a single compound and the polarographic curves are suitable for analytical determinations (564). A colorimetric method was used for the determination of methylhydrazine (298). The reagent, p-dimethylaminobenzaldehyde in aqueous sulfuric acid, is also employed for the determination of hydrazine and gives slight coloration n-ith urea and adrenalone. The extinction is measured at 458 mp. Morise has reviewed the French Codex method for the determination of 7naminophenol in sodium PAS [&aminosalicylate] (327). Knobloch (264) and also Coulson and Hales (105) reported a n infrared procedure for mixtures of cuJp,~-picoline,2,6,-lutidineJ and 2-ethyl pyridine in the @-picoline fraction of pyridine base. The infrared spectra of quinoline and isoquinoline \vere observed in carbon disulfide solution and theii use in industrial analysis IT-as studied (250). Quantitative analysis of pyridine bases from coal tar were analyzed by ultraviolet and infrared absorption spectra (17jl 513, 515). TKOdimensional paper chromatograms of pyridine and piperidinecar boxylic acids were described by Hulme (224). The positions of the compounds were revealed by spraying with conventional reagents. R, values olitained in several solvent systems were reported. A precise and accurate method for the polarographic determination of 2-cyanopyridine was described (230). Experimental conditions for the determination of pyridine by use of the reaction with copper and thiocyanate were described (331). The reduction of nitroguanidine by titanium(II1) chloride in hydrochloric acid \vas studied (65). ANTIBIOTICS
Ashton and Foster (11) report a specific method for benzylpenicillin by the isotope dilution technique using carbon-14 benzylpenicillin. The penicillin is precipitated as the S-ethylpiperidine salt. Penicillin G reacts with Fehling’s solution and releases ammonia (508),which is determined in the conventional manner. Three methods are available for determining the penicillin content of ointments-namely, extraction and iodometric assayJ extraction and back-titration of iodine, and extraction, nitration, and colorimetric determination (338). Penicillin V absorbs iodine after inactivation with penicillinase and has a molecular extinction coefficient of 1330 a t 268 mp and 1100 a t 274 mp, and develops a deep blue-purple color in sulfuric acid with chromotropic acid when heated in a glycerol bath a t 105” C. (3.59). The greater stability of penicillin V in the iodometric assay R-as used as the basis for assay of penicillin V in the presence of G (181). Penicillin V can VOL. 29, NO. 4, APRIL 1957
701
be separated from G or K by paper chromatography (480). Pan (357) determined o-oxyphenylacetic acid in penicillin fermentation broth by nitration. N , N - Dibenzylethylenediamine dibenzylpenicillin (263) lends itself to titration in acetic acid with perchloric acid (crystal violet indicator). Neomycin B and C can be separated by paper chromatography after acetylation (368). Actinomycin C has an analytically useful half-wave potential in several systems (37) and can be separated and identified by paper chromatography (528). A method for dehydrostreptomycin is based on the precipitation of the Reineckate (ammonium tetrathio cyanodiammonochromate), separation, hydrolysis with Fehling’s I1 solution, acidification with nitric acid, and then back-titration of an excess of silver nitrate with ammonium thiocyanate solution (530). Banerjee (t0)has analyzed streptomycin and dehydrostreptomycin colorimetrically using the Voges-Proskauer reaction. Colorimetric methods for dehydrostreptomycin based on reaction with sodium pentacyanoamminoferroate, potassium ferrocyanide (%$4), and sodium nitroprusside (523) have been reported. Alkaline hydrolysis of erythromycin produces a material with characteristic ultraviolet absorption (600). Erythromycin in ethyl methyl ketone develops a color with methyl sulfate Kith a maximum at 480 mM; Beer’s law is obeyed between 50 and 300 y (371). Terramycin when treated with hydrochloric acid and sodium nitrite forms a nitroso compound whose half-wave potential in an ammonia-ammonium chloride buffer solution is proportional to the concentration (341). The tetracyclines have been separated and identified by paper chromatography, using an ascending technique and a mixture of formic acid and the separated organic phase from a mixture of n-butyl acetate, isobutyl methyl ketone, 1butanol, and water (161). Identification reactions have been reported by Laubie (280). The tetracyclines can be separated by countercurrent distribution, using 50 tubes, in the system sodium orthophosphate (hTazHP04)-citrate p H 4.5 us. chloroform: chlortetracycline peaks a t 26, tetracycline 39, and oxytetracycline 44 (323). Tetracycline can be determined spectrophotometrically in the presence of chlortetracycline; the method is based upon changes in absorption in acid and basic media (652). Tetracycline reacts with ammonium molybdate solution (246). Aureomycin develops a color with phosphomolybdic acid under controlled conditions (389), and complexes with thorium(IV) with a maximum a t 405 mp (426). Derivative polarography has been
702
ANALYTICAL CHEMISTRY
applied t o the determination of chloramphenicol (265),which is also measured colorimetrically with dimethylformamide in the presence of tetraethylammonium hydroxide and acetone (170). Cycloserine reacts with sodium nitritopentacyanoferroate in slightly acid medium to give an intense blue color (238). Ashton and Brown (10) have studied the analysis of griseofulvin and have reported the spectrophotometric properties; while Ashton ( 9 ) described an isotope dilution method using chlorine-36. CHEMOTHERAPEUTICS
0
Os01 and Sideri (353) have reported a scheme for the identification and differentiation of 13 U. S. Pharmacopoeia antihistamines based upon the color in sulfuric acid and the change upon adding water. The ultraviolet absorption spectra of eight antihistamines, such as Antazoline hydrochloride and Pyribenzamine hydrochloride, in water a t different p H values and in alcohol have been listed (262). Other papers (49, 53) reported the ion exchange and spectrophotometric assay of ten antihistamines. Mixtures of pyrrobutamine diphosphate and methapyrilene hydrochloride can be assayed spectrophotometrically (52). Paper chromatographic separation (76) and color reactions (337) for antihistamines were reported. Nupercaine and derivatives have been studied polarographically and a system for paper chromatography was reNitrogen-containing ported (522). bases such as procaine and antipyrene have been titrated with phosphotungstic acid using a dropping mercury electrode (667). Identification procedures and a colorimetric assay have been reported for n-butyl-aminoacet-6-chloro2-methylanilide hydrochloride (203). Local anesthetics containing an alkylthio group develop color with selenious acid in sulfuric acid (340). Paper chromatography procedures for local anesthetics were reported (390), as well as toxicological methods (33). The hydrolysis of procaine can be followed spectrophotometrically (399). Aspirin, phenacetin, and caffeine can be determined by anhydrous titration methods. The aspirin is titrated in dimethylformamide with lithium methylate in benzene-methanol using thymol blue. Phenacetin is titrated with perchloric acid in p-dioxane using glasscalomel electrodes, and the caffeine is treated with iodine and back-titrated with sodium thiosulfate (561). Partitioning of aspirin, phenacetin, and caffeine using chloroform and sodium bicarbonate separates the compounds, which are then determined colorimetrically (359). The decomposition of aspirin in aspirin, phenacetin, and caffeine has
been shown to depend upon the lubricant used in tableting (396). Salicylic acid can be determined fluorometrically (511). p-Acetophenetidine can be determined by hydrolysis, diazotization, and coupling (18). Meperidine hydrochloride can be determined in alkali using bromosulfthalein (299). Pyramidon can be coupled with diazotized nitroaniline (202) and Veramon is titrated with p-toluenesulfonic acid using dimethylaminoazobenzene as indicator (377). Aminopyrine, isonicotinic acid hydrazide, and hexamethylentetranline can be determined by indirect complexometric methods (79). Rotodaro (419) reported an extraction procedure for barbiturates in pharmaceuticals. Several paper chromatographic procedures have been reported (188, 223, 283). Canback (80) tabulated the infrared spectra of Nujol mulls of the barbiturates. Ultraviolet spectrophotometric methods were reported (58, ,299, 303). A procedure for the sublimation of barbiturate and its identification crystallographically was listed (74). Barbituric acid derivatives produce an intense red-violet color with copper-pyridine reagent (266). Barbiturates can be titrated in a chloroform-polyethylene glycol 400 solvent system using sodium methoxide in methanol as titrant (488). Color reactions of thiobarbituric acid derivatives were reported (209) as well as quantitative methods (549) for their determination. Priscoline can be determined gravimetrically by precipitation with Reinecke’s salt or tungstosilicic acid (54). Privine (2 - naphthyl - 1 - methylimidazoline) was precipitated with picric acid and the precipitate was determined polarographically (342). Solvent systems have been reported for the identification of 20 imidazole compounds by paper chromatography (106). Imidazole compounds and histidine form, with p-nitrobenzoyl chloride in an alkaline medium, colored solutions which are specific (107). A method was described for the determination of adrenaline and noradrenaline in urine based on absorption on alunlina at p H 8.5, elution with oxalic acid, and measurement of the fluorescence after manganese dioxide oxidation (568). Iodate oxidation of noradrenaline and adrenaline provide polarographically assayable iodo- derivatives (210). Feigl and Feigl (164) reported a selective method for ephedrine and adrenaline. Assay of ephedrine in tablets of aminophylline and sodium phenobarbitone is based on a conventional technique (46). Electrophoresis (361) and partition chromatography (424) have been suggested for the separation of sulfonamides. A bromometric method (254) and a colorimetric method (535) have been reported, The first half-wave potential (-0.19 volt us. the S.C.E.) of
phenylmercuric acetate is independent of the pH value, but the second wave (- 1.14 volts a t pH 10) is pH-dependent. The m v e height of both waves is proportional to the concentration nithin the range of 1 to 10 X 10-4M in 0.1M potassium nitrate (243). Spectrophotometric methods for chlorocresol in injectables has been reported (66). Heyylresorcinol can be determined colorornetrically (64). Isonicotinic acid hydrazide and nicotinic acid in the presence of p-aminosalicylic acid and m-aminophenol can be separated by countercurrent distribution using 2butanol and water as the phases (565). Electrophoretic and paper chromatographic methods are reported for isonicotinic acid hydrazide (113). The hydrazine group in isoniazide reduces mercury potassium iodide a t p H > 10; upon acidification the solution turns yellowish green and is measured colori(538). Compleximetric metrically methods are reported for hexamine and isoniazide (77). m-Aminophenol in paminosalicylic acid can be determined by the color reaction with 4-aminoantipyrine in the presence of potassium ferricyanide (165). The blood level determination of p-aminosalicylic acid and isoniazide with vanillin a t a controlled pH was reported (122). It was reported that insulin can be controlled by circular-paper chromatography (356)) polarography (531), and reaction with ninhydrin (479). I n mer curial diuretics where mercury is loosely bound to carbon, the mercury can be converted t o titratable mercury by dissolving in 50% sulfuric acid followed by titration with potassium thiocyanate (501). Terpin hydrate can be determined spectrophotometrically by treating with phosphomolybdic acid (376). Anion exchange resin (Dowex2) can be used in the isolation and quantitative estimation of iodide, thyroxine, and substances related to thyroxine (48). Warfarin treated with an alkaline solution of iodine yields iodoform. The precipitate can be determined polarographically ($70). Santonin can be determined colorinietrically (655) or by paper chromatography (324). GLYCOSIDES
The utilization of chromatography in the identification, separation, and determination of glycosides continues to receive increasing prominence. Rohatgi (406) identified digitoxin and digoxin from their acetyl compounds by paper chromatography, using propylene glycol or formamide as the stationary phase and benzene-chloroform as the mobile phase. Raymond’s reaction, based on the genin portion of the glycoside, was employed to develop the spots. A qualitative and quantitative study of aloe, aloin, and cascara Sagrada by
paper chromatography has been reported (311). The separation and determination of individual lanatosides and diacetyllanatosides in lanatoside ABC was achieved by paper chromatography (222). An ethanolic solution of glycosides was placed on paper and then saturated with formamide solution. The developing was carried out with chloroform or chloroform-ethyl acetate. The separation was observed by ultraviolet light and spots were developed with xanthydrol reagent; the absorbance was measured colorimetrically. Additional paper chromatography techniques have been reported ($42, 251, 438, &9), and the paper partition chromatography of digitalis purpurea glycosides has been described (433). As a supplement to a prior paper, the separation of purpurea glycosides A and B of digitoxin, gitoxin, and diginin, using reverse-phase chromatography, was reported by Gunzel and Weiss (189). Anisaldehyde-acetic acid-sulfuric acid reagent was used to identify the bands. A chemical method for the determination of digitalis activity has been described by Kolfgramm and Weiss (550)and a colorimetric technique for digitoxose has been reported (124). METALLIC IONS AND SIMPLE ANIONS
It is questonable whether a section on metallic ions and simple anions belongs in a review of pharmaceutical analysis-however, other reviews do not exactly approach this type of analysis with the sanie viewpoint, and these anions and cations are an integral part of pharmaceutical analysis. Trace impurities in drugs may have a marked effect on the activity and stability of the product. The blue color obtained when sodium phenate is added to a solution of ammonia that has been treated with hypochlorous acid forms the basis of a method for the determination of ammonia (439). A new four-component system has been devised (isopropyl alcohol, pyridine, acetic acid, and water) for the paper chromatography of alkali and alkaline earth cations (183). Higuchi and Rehm (216) have titrated sulfuric acid and hydrochloric acid in anhydrous acetic acid with alkali acetates. The results present for the first time the neutralization end points of the two hydrogens of sulfuric acid in acetic acid. Under suitable conditions oxalic acid and indole react t o form a red or pink colored compound which conforms to Beer’s law (40). An aniperometric method using rotating platinum or gold electrodes has been used to determine dissolved oxygen (6441). Laitinen and Woerner (276) applied the amperometric titration method to arsenite, ammonia, and thiocyanate,
using hypochlorite in the presence of bromide. An extremely sensitive reaction for chloride, bromide, iodide, and sulfide has been published by Kirsten. These ions in a solution containing phosphoric acid, ethyl acetate, and chloroform react with the silver of the diphenylthiocarbazone silver complex. Diphenylthiocarbazone is liberated and measured a t 598 mp (261). Potentiometric micro methods have been reported (102,168,222,227,287,275). The diffusion technique, absorbing the chloride in sodium arsenate (NaAs02) was reported (334). A differential spectrophotometric method for fluoride (191) using thorium-Alizarin Red S reagent and a gravimetric method as calcium fluoride were reported (16). A modification of the U. S. Pharmacopoeia X I V procedure for Vioform through the determination of the chloride and iodide content has been published (527). Methods of assay of sodium iodide-131 have been described (575). Cuprous iodide is insoluble in in 0.1N sulfuric acid which serves as a gravimetric method (175) for its determination. By means of ascorbic acid, using a two-stage titration under controlled acidity, iodine and iodide were determined (lab). The action of iodine on Variamine blue gives a blue product useful for determining iodine (147). Cyanide ( 5 y) can be determined in the presence of large amounts (2.5 mg.) of sulfide with an accuracy within 5570, using benzidine as the reagent (17). A spot test which detected as little as 1 y of cyanide was reported (153). From 1 to 40 y of nitrite in 0.19 hydrochloric acid can be determined n ith sodium naphthionate (59). A Iijeldahl method for the determination of microgram quantities of nitrogen by reaction of the formed ammonia in a suitably buffered solution with sodium hypobromite and measurement of the excess reagent by titration of the iodine liberated by it from acidified potassium iodide has been described (130). A two-step mixed indicator for Kjeldahl nitrogen titration has been proposed (463). The popularity of ethylenediaminetetraacetic acid (EDTA) as a coniplexing agent is increasing rapidly (71). Patton and Reeder (365) used 2-hydroxy 1-(2hydroxy - 4 - sulfo - 1 - nnphthg1azo)3-naphthoic acid for the determination of calcium in the presence of magnesium .rr-ith EDTA, whereas Lliehl and Ellingboe (129) suggested calcein as the indicator. A method for the simultaneous spectrophotometric determination of calcium and magnesium with Eriochrome Black T as indicator has been reported (560). Abbott and Reber ( 1 ) used a cation exchange resin to convert magnesium citrate to citric acid and determined magnesium by titration n.ith EDT.4. Zirconium in acid solution can VOL. 2 9 , NO. 4, APRIL 1957
703
be determined by addition of an excess of EDTA and back-titration (173), or spectrophotometrically (322). A new color reaction for iron and cobalt has been reported using EDTA and nitrilotriacetic acid (91). Milligram quantities of barium can be determined spectrophotometrically using Eriochrome Black T as the indicator (420),and the absorption of bismuth EDTA complex x a s found suitable for quantitative determination (544). Other complexometric methods were reported for boric acid with tartaric acid (453), zirconium with Versene (19), or Alizarin Red S (306). Methods have been reported for the determination of the following: cobalt with 1-nitroso-2-naphthol (429); calcium with alizarin (332); iron with 4 7 - dehydroxy - 1 , l O - phenanthroline (440) and with 5sulfanthranilic acid (665); magnesium with thiazole yellow (268); decarborane with Coramine (9N-diethylnicotinamide) (217 ); iron(111)with 4-amino-4’-methoxydiphenylamine (146). Siggia (466) reported the potentiometric titrations of metal ions involving chelating agents. Iodoxine (8-hydroxy5,7-diiodoquinoline) reacts with copper, nickel, cobalt, iron, vanadium, lead, thorium, zirconium, and titanium to give different colored precipitates which are useful for identification (330). Cupric ion can be determined using salicylic acid and nitrite (519) and traces (0.02 y) of copper in water can be determined by using diethyldithiocarbamate (271). Iron has been determined by the ultraviolet absorption of ferric perchlorate (S2). Iron was also determined colorimetrically after exchanging for iodate ion with granular silver iodate in a column. The released iodate reacts n-ith cadmium iodide which is measured a t 615 mp with starch (277). A simplified dithiocarbamate method for as little as 1 p.p.m. of lead in food was reported (176). A great number of papers have appeared on the determination of mercury, a few of which are of particular interest to pharmaceutical analysts. The mercury content of 3-chloromercury-Zmethoxypropylcarbamide in tablets has been determined by amalgamation with zinc after oxidation Lyith bromine (41). A quick method for the microdetermination of mercury in organic substances in the presence of halogen depends upon ashing in calcium oxide, acidifying, and titrating with ammonium thiocyanate solution (436). FJ‘alton and Smith decomposed the organic compound with hydrogen iodide and iodine (HI+12). Mercury formed the Hg14-- ion which was precipitated and weighed as cupric propylenediamine mercuric iodide (639). A procedure for preservatives containing mercury in beverages was listed (140). A rapid microprocedure for mercury in biological material requires
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ANALYTICAL CHEMISTRY
a sulfuric acid-hydrogen peroxide digestion and measurement colorimetrically with dithizone (361, 381). A rapid and precise measurement of moisturein nonvolatile biologicals,foods, and pharmaceuticals has been reported by Heckley (208). Moisture is evaporated from the sample under high vacuum and condensed in dry ice and ethyl alcohol. The moisture is then volatized and the pressure is measured. The method is possibly good for as little as 0.01 mg. of water. In addition, the apparatus may be used to determine the internal pressure of ampoules and vials. A gain in stability of the Karl Fischer reagent was obtained by substituting methyl Cellosolve for methanol (372). A patent has been issued on the spectroscopic determination of total water content with heavy water (70). STEROIDS
Hydrocortisone in a mixture of sulfuric acid and glacial acetic acid produces a yellow color with a maximum a t 470 mp, whereas cortisone does not produce a color in this reagent. This provides a method which is useful for the identification and determination of hydrocortisone in substance, in formulations, and in mixture with cortisone (493). A microchemical detection of the characteristic functional groups in the steroid side chains, using a series of reagents, has been devised (14). Phenolic steroids can be determined using perchloric acid and picric or salicylic acid (382). The ultraviolet absorption spectra of 34 steroids of the urinary and cortical series in “100”% phosphoric acid were listed. It is claimed that the color is more reproducible and stable than nhen sulfuric acid is used (343). Colorimetric methods for steroids in urine were reported (55, 89, 178, 211, 431, 470). Testosterone propionate and progesterone in oil solutions can be assayed colorimetrically using isonicotinic acid hydrazide (521). Total adrenal cholesterol forms a purple color in a mixture of concentrated sulfuric acid, glacial acetic acid, and ferric chloride (421). A series of color reactions of steroids has been studied (383). Ergosterol in fermentation broth may be analyzed by the Liebermann-Burchard reagent (482). A procedure was described for the determination of desoxycholic acid and cholic acid by heating in 65% sulfuric acid and measuring the absorption a t 320 and 385 mp (328); the benzaldehyde method was improved by mixing benzaldehyde and sulfuric acid prior t o addition to the sample (278). Cholic, desoxycholic, and dehydrocholic acids were titrated potentiometrically by nonaqueous means (187). Meyer and Lindberg (321) have studied the characteristics of hydroxylated and ketonic
steroids to&ard blue tetrazolium. Diethylstilbestrol reacts with bromine to form a colored complex which has a maximum a t 500 mp (132). A simplified distilling head for the measurement of corticosteroids by the formaldehydogenic procedure has been proposed (196). Sodium borohydride has been suggested for the determination of 3-ketobisnor-4-cholene-22-al (233)* I n line with the current vogue, many papers have been published on the paper chromatographic separation of steroids. Testosterone, isoergosterone. and A-1,4androstadiene-3,20-dionehave been separated (56) as well as adenosinetriphosphate (87), the estrogens (308, 386, 417), corticosteroids (IS’S), and a-ketolic steroids (509). The estrogens in urea have been determined by fluorescence (295), as well as A4-3-ketosteroids (Z), and chlorsterol (4). A workable and reliable isotope dilution assay for compound S and compound F in fermentation liquors has been reported. A total of seven steroids were deuterized for use. Compounds S and F were separated by countercurrent distribution and purified. The atom per cent deuterium was determined from the infrared absorption of the water of combustion a t 3.98 microns (241). Adrenochrome seniicarbazone in 0.5N sodium hydroxide with 0.5% sodium sulfite and 1 t o 8 drops of a saturated alcoholic methyl red solution for each 20 ml. has a half wave of -0.71 volt (469). A buffer at p H 8.5 with an alcohol content of 90% has been found to facilitate polarography of Cls-,C21-, and &-keto steroids (400). The infrared spectrum of isosapogenin acetates (139), steroids with reduced ring il (Ql8), adIenocortica1 hormones (205), and the influence of the solvent on carbonyl frequency have been reported (497). Six corticosteroids have been separated by 24 to 48 countercurrent transfers using nine different solvent systems (86). Testosterone, cortisone, and desoxycorticosterone have been separated and determined by paper electrophoresis (460). X-ray diffraction data on 39 steroids (362) and crystallographic data for hydrocortisone acetate (462) have been reported. SUGARS A N D CARBOHYDRATES
A colorimetric method for the determination of invert sugar in the presence of sucrose is based on reaction with triphenyltetrazolium chloride (555). Forist and Speck reported the oxidimetric determination of glucose, xylose, ribose, and glyceraldehyde by means of ceric perchlorate reagent (164). By use of a phenol-sulfuric acid reaction, a method has been developed to determine submicro amounts of sugars and related
substances (153). I n conjunction with paper partition chromatography the method is useful for the determination of polysaccharides and their methyl derivatives. The method is simple, rapid, sensitive, and gives reproducible results. The separation of mannose from a mixture of sugars has been determined by a paper chromatographic procedure (273). Mannose is separated on Whatman KO.1 filter paper, buffered a t p H 2.0 using phenol, 1-butanol, glacial acetic acid, and water. The sugar spots are developed with benzidine trichloroacetic acid reagent and heat. The method is also applicable for the identification of arabinose. A colorimetric technique for the determination of sugars m-ith 3,4-dinitrobenzoic acid n a s described in a Japanese journal (494). The extinction of the yellow chromogen obtained with the reagent was measured a t 395 mp. A modification of the colorinietric method of .lminoff for the estimation of N-acetylamine sugars has been reported (394). Sugar alcohols and their glycosides have l)een detected by treatment with p nnisidine after paper chromatographic separation using a mixture of 1-propanol, ethyl acetate, and v-ater (86). The Rz values for 19 polyols are listed. A new spray technique for the detection of sugars on paper chromatograms has been described. The reaction involves the reduction of alkaline picrate to picramic acid or other reddish spots (390)). The reaction of sulfonyl hydrazides rvith sugars is the basis of an identification technique for monosaccharides (546). crystalline compounds were obtained by heating the components in methyl cyanide a t SO" for 10 to 15 minutes. Polarographic behavior of saccharin in various solutions was described in a Russian journal (336). The determination is satisfactory in either 0.1.V potassium chloride or 0.liZ' hydrochloric acid. The use of lead tetraacetate oxidation for determining the structure of reducing disaccharides has been reported ($69). Mixtures of monosaccharides have been separated by twodimensional ascending paper chromatography (197) using a mixture of 1butanol, pyridine, and water in the first dimension, and phenol saturated with water in the second dimension. 2-Kaphthylamine was used to detect the sugars. Methods for the detection and estiniation of glucosamine and galactosamine have been described (169, 409). The latter method is based on treatment with acetylacetone and Ehrlich's reagent. The former utilizes paper chromatography to separate them one from another and from other saccharides. Quantitative estimation is based on reaction with ninhydrin or by reduction of triphenyltetrazolium hydroxide. I n
the examination of parenteral solutions of glucose by ultraviolet absorption, Iwamoto, Saito, and Taga found 5hydroxymethylfurfural and another substance not definitely identified (128). Nine components in corn sirups were separated and identified by descending paper chromatography (547). The solvent most successfully employed was a mixture of butanol, ethyl alcohol, and water. The spots were identified by an aniline acid phthalate spray and subsequently mere determined colorimetrically n i t h phenolsulfuric acid reagent a t 490 mp or alkaline dinitrosalicylic acidphenol reagent a t 543 mp. A relatively specific colorimetric technique for the determination of 5-ketogluconate is based on reaction n-ith l-methyl-lphenylhydrazine sulfate (449). The components are buffered t o p H 3 t o 4 and heated at 98" C.; the resultant rosecolored solution is measured at 350 mp. Alt (6) described a spectrophotometric method for the assay of gluconic acid and its salts based on the color deyeloped by a copper-gluconate complex in alkaline solution. The microscopic identification of microgram quantities of arabinose and fructose by a solvent diffusion technique has been described (458). Schoch and RIayrdcl (448)examined modified starches by a microscopic technique based on the observation that positively charged dyes stain anionic products, such as oxidized starches and carboxymethyl ethers, vhereas cationic starches are stained only by negatively charged dyes. Carbohydrates 11-ere determined by oxidation with ceric sulfate under reflux and subsequent hack-titration of excess ceric sulfate (461). Diacetone sorbose has been separated from the mono compound and condensation products of acetone by countercurrent distribution using a benzene-n ater system (252), VITAMINS
Rogers (404) has prepared a table for the rapid application of the MortonStubbs correction in vitamin A assay. An ultraviolet method has been reported for the determination of vitamin A and carotene in butter (467). The anion exchange resin Duolite A2 absorbs vitamin A in petroleum ether and it is eluted with protophilic solvents (339). Determinations of p-carotene in vitaminized chocolate (S9),in plant material ( I S ) , and in dehydrated alfalfa meal treated with iV,N'-diphenyl-p-phenylenediamine (247) have been reported. The ultraviolet absorption of vitamin B, shows an isobestic point a t 273 mp over the p H range 1 to 9 (f3f). Ohnesorge and Rogers (350) utilized the differences in the fluorescence spectra of thiamine and riboflavin to measure each in the presence of the other. This
method has the desired accuracy. A polarographic method for determination of thiamine in the presence of ascorbic acid has been described (464). A method based on the hydrolysis of thiamine to ammonia has been suggested (422),aswellasa method for determining the riboflavin content of sweet potatoes (667). Separations of the B complex vitamins by electrophoresis on agar plates (309) and paper chromatography (179, 403) were reported. VanXlelle has published a method for the determination of the vitamin B12-likematerials in liver injection by purification through a cation eychange resin (626). The method is excellent for those products known to be free of red pigments. An eight-tube countercurrent distribution of vitamin B12using water saturated with benzyl alcohol, and benzyl alcohol saturated with water, as the phases has been published. This method separates the red pigments from the cyanocobalamin or cobalamins convertible to cyanocobalamin (SOW), A modification of the dicyano complex procedure has been reported (82). Sullivan and Clarke (486) have reported a highly specific procedure for ascorbic acid. The method depends on the reduction of ferric chloride by ascorbic acid and the colorimetric determination of the ferrous chloride by means of the reaction of 2,2'-bipydridine. A supplemental paper has appeared on the diazotized 4-niethoxy-2-nitroaniline method in which the method has been applied to certain foods and biological fluids ( & I ) , Ascorbic acid is selectively oxidized by iY-bromosuccinimide (66). Paper chromatographic methods (235, 444) have been reported as well as a method for determining ascorbic acid in flour ($06))a colorimetric procedure, (4,963, an iodometric method using platinum tungsten electrodes (366),and a procedure to determine reduced and unchanged ascoibic acid in biological material (435). Lyness and Quackenbush (293) made vitamin D2 and Da react with iodine in ethylene dichloride. The intensity of the color was enhanced by mercury-p-chlorobenzoate. The reagent showed high specificity for vitamin D. Furfural and sulfuric acid give a colored reaction product with D2 and DS (281). Stannous chloride in acetyl chloride produces a useful color (5f2). Calciferol has been determined colorimetrically (75). A gravimetric method for nicotinamide has been proposed (50), as n-ell as a polarographic method (88). GENERAL
Warner and Raptis (640)have described the determination of formic acid in the presence of acetic acid by azeotropic distillation with chloroform and subsequent titration of formic acid VOL. 2 9 , NO. 4, APRIL 1957
705
with standard base. Infrared evaluation of sodium salts of organic acids has been reported (94). The spectral absorption bands of the salts show a marked degree of specificity in contrast to the acids themselves. The determination of adipic acid and adipicglutaric acid mixtures was described in detail. A titrimetric method of assay for trichloroacetate is based on decarboxylation (447). Two direct methods for acetic acid in acetic anhydride depend on reaction with a tertiary amine (236). I n the first procedure the sample is titrated with triethylamine to a methyl red end point. I n the second procedure, the temperature rise hen the reagent is added to the sample is noted and the concentration of the acid is ascertained from an empirical curve. A nen- method for the determination of citric acid (63) and a n adaptation of the pentabromoacetone technique for citric acid have been published (433). The former is based on the formation of a soluble copper complex and is suitable for mixtures using aqueous ethanolic ammonia for the chromatography. I n the latter technique, the converted pentabromoacetone is determined colorimetrically by its reaction with sodium sulfide. Methods for the determination of malic acid in plant material and wine have been reported (329, 393). Chromatography has been utilized in the separation of organic acids (395). Scott (456)employed ketones in place of alcohols in combination with halogenated hydrocarbons as solvents; a new solvent system, methylcellulosewater and Skellysolve B, for separating monocarboxylic acids and dicarboxylic acids was reported (562). 2,6-Dichlorophenol-indophenol has been proposed as a spray reagent in the paper chromatography of organic acids (23). A chromatographic determination of carboxyl groups in filter paper makes use of the bond of metal ions to the carboxyl groups (518). A system for paper chromatography of bile acids was described by Kritchevsky and McCandless (974). Blpha-keto acids were isolated as 2,4-diiiitrophenylhydrazones and separated by paper electrophoresis (498). The separate bands were extracted with caustic and color intensities were measured colorimetrically. Determination of carboxylic acid anhydrides by reaction with morpholine and titration of the excess reagent with methanolic hydrochloric acid using methyl yello\vmethylene blue indicator rias reported (236). The identification of benzoic acid, p-chlorobenzoic acid, and p-hydroxybenzoic acid in cheese has been described by Jarczynski and Kiermeier (999). Details for chromatographic separation were outlined: confirmation of benzoic acid and pchlorobenzoic acid was by Lfohler’s
706
ANALYTICAL CHEMISTRY
reaction and p-hydroxybenzoic acid was detected with diazotized sulfanilic acid. Another technique for the detection of benzoic acid by hydroxylation is based on the development of a violet color with iron(I1) (408). The thioindoxyl reaction for monohaloacetic acid has been employed for the detection of fluoroacetic acid in foods (957). Utilizing a diffusion technique, Stark (478) reported a method for the determination of lactic acid. The lactic acid nas oxidized by ceric sulfate to acetaldehyde, which was collected in thiosemicarbazide solution in a special diffusion apparatus; the thiosemicarbazone formed n as determined by measuring the estinction a t 261.5 mp. A semispecific method for the detection of sorbic acid, a nen preservative, has been reported (141). The sample was oxidized to acetaldehyde ith permanganate, separated from salicylic acid by distillation, and made to react with Schiff’s reagent. Negative results were obtained with other preservatives, but a positive result mas obtained n i t h cinnamic acid. The scope of nonaqueous titrimetry of organic bases and acids continues to grow. A comprehensive review and brief account of the theory of titration of weak acids in nonaqueous media lvere given by Backe-Hansen (15). High-frequency titration of acids (118) and bases, phenols and enols (273) in dimethylformamide, glacial acetic acid, and ethylenediamine, respectively, has been discussed. Several potentiometric techniques for the determination of weak acids and bases have been published, including details of various electrode systems, nonaqueous media, and titrants (36, 11’7, 186, 253, 504). Tetrabutylammonium hydroxide (112) and diphenyl phosphate (115) have been used as titrants for acids and bases in nonaqueous solvents. Smith (471) reported the preparation and standardization of perchloratoceric acid solution in perchloric acid. -4s it is stable for a limited time, storage in the refrigerator is necessary. The titrant is standardized against sodium oxalate using nitroferroin as indicator. Phenol-chloroform-acetonitrile (90) and acrylonitrile (356) {Tere employed as the solvent systems in nonaqueous titrimetry. Pyridine, diphenylguanidine, triethylamine, and benzylamine n ere titrated coulometrically \$ith lithium perchlorate trihydrate (483). ilromatic amines are not titratable by this procedure. Picric acid has bren assayed by coulometry a t a controlled potential. the reduction proceeding quantitatively to 2,4,6-triaminophenol(S19). Tungstosilic, tungstophosphoric, and molybdophosphoric acids were found suitable for the polarographic titrations of organic bases (475). A cation exchange resin as used to convert alkali salts of organic acidsLe., sodium citrate, sodium salicylate,
etc.-to the free acid and this was followed by direct titration n ith sodium hydroxide (51). The salicylaldehyde method for pyruvic acid has been studied and modified by Berntsson (43). A colorimetric method for organic bases based on reaction n ith hexanitrodiphenylamine to form a red colored anion has been reported (259). Details for pyridine in alcohol n ere described. Data showing the effect of organic bases on the partition of indicators in a chloroform-n ater system and application of the effects to titration of bases vc‘ere shon-n (507). ,4 new method for the colorimetric determination of formaldehyde (184) and a neiv spot test for formaldehyde ( 5 4 ) have been described. I n the former, the reagent is prepared by dissolving sodium sulfite in a solution of methyl violet containing hydrochloric acid. A color is formed nith formaldehyde which has an absorption maximum a t 578 mp. The spot test is based on the addition of formaldehyde to an equilibrium mixture of potassium tetracyanonickelate and dimethylglyoxime. Cyanide ion is removed through the formation of cyanohydrin with the subsequent liberation of nickel(I1) and the formation of the red nickel dimethylgloximate. A technique for the determination of formaldehyde is based on oxidation of the phenylhydrazone of formaldehyde n ith potassium ferricyanide (354). The color is developed by the addition of hydrochloric acid, extracted quantitatively with butanol, and the absorption is measured a t 520 mp. A method based on the oxidation of aldehydes to acids by niercury ion was described (463). The reduced free mercury was determined iodometrically. Farr (150) reviewed the analytical methods for the determination of aldehydes and ketones: (1) bisulfites, (2) ru’essler’s reagent, (3) oxidation of formaldehyde, (4) argentimetric methods, and ( 5 ) hypoiodites. A new technique, using peroxytrifluoroacetic acid for the determination of the carbonyl function has been reported (904). The sample n a s treated with excess reagent and residual peroxy acid was determined iodometrically. A study confirmed the suitability of anhydrous methanol as a solvent for polarographic reduction of carbonyl compounds (405). I n a new method glyoxal nas determined as its Schiff’s base n ith cyclohexylamine (34). A spectrophotometric method for the determination of glyoxal is based on absorbance of the 2,i-dinitrophenylhydrazone derivative in alkaline acetone a t 600 mp (26). A similar technique for carbonyl compounds n as outlined by Yamaguchi (556). The polarographic determination of aldehydes as the 2,4-dinitrophenylhydrazone has been described (373). The reaction of alde-
hydes ith unsj-ninietrical dimethylhydrazine n-as used as the basis of a volumetric technique. Excess dimethylhydrazine was added to the sample and, after reaction, the excess was titrated with standard acid (468). Ketones cannot be determined by this method. Using phenoxyethanol-impregnated paper as the stationary phase and heptane as the mobile phase. good separation of the 2,4-dinitrophenylhydrazones of carbonyl compounds n as obtained (294). Color reactions of aldehydes and ketones n i t h alkaline-vanillin reagent have been published (281). The experimental cwnditions for a color reaction between acetone and 2-naphthol esters have 1)een described (407). The deteimination of carboxyl compounds is based on ovimation n ith hydro tate-acetic acid (214). The oximes formed are titrated IT ith perchloric acid-glacial acetic acid. Improved methods of tests for alcohols, chloral, salicyl alcohol, salicin, sulfoxylated compounds, and some organic reducing agents have been described ( I C 9 ) . Vanadium 8-quinolinolate as a reagent for the detection of alcohols. thiols, and amines was reported ( 4 7 ) . The polarographic detection of ethyl alcohol is based on oxidation to acetaldehyde n hich is then de(445). termined polarographically Conventional techniques for the detection or determination of higher alcohols in distilled spirits were described and compared (318). Dinitrophenylamine alcohol has been assayed, using silica gel-Celite columns (183). The colorimetric determination of trace amounts of alcohols utilizes ceric ammonium nitrate as a reagent (392). Spanyer and Phillips separated C1 to Cs alcohols as the potassium xanthates on cellulose columns (476). A method has been presented for the determination of as little as 50 y of alcohol in 20,000 parts of i r ater 11 ith an accuracy within rt 17(634). A modification of an isotope dilution nietliotl n as proposed for the determination nf hydroxy and amino compounds (4'74). The compounds are first converted to a chlorine-containing clerir ative and then determined by a convcntional isotope dilution method. Thii technique has been applied to the detc,rmination of phenol, pyrocatechol, nrethanol, ethylene glycol, aniline, and ethylenediamine. Polarographic reduction 11aves of a number of saturated acids and unsaturated fatty acids that have double bonds in other than a-P position were observed in aqueous acetone containing lithium chloride as the supporting electrolyte (510). de Castro and Jannke (119) demonstrated t h a t chromatographic procedures can be applied successfully in the total analysis of a natural fat. Methods for the separation of fatty acids by paper chromatog-
raphy have been described (2~55,266, 434, 537). A partition chromatographic technique has been reported for the separation and determination of straight-chain carboxylic acids (103) using as the stationary phase 2;M aqueous glycine on silicic acid and as the mobile phase a mixture of butanol and chloroform. O'Connor (345) revieir ed the application of infrared spectrophotometry to cis, trans isomerism, autoxidation, rancidity, polymerization, molecular n eight, and other miscellaneous problems. The value of mass spectrometric analysis for both qualitative and quantitative determinations has been demonstrated (300). The mass spectra of glycerol, 2,2'-dipyridylamine, nicotine, and many other compounds werp shown. A method for analysis and identification of oils by examination of their unsaponifiable matter !vas described (12). LIethotls for the determination of the color of oils have been discussed; typical light absorption curves of raw and refined oils n ere illustrated (533). The polarographic beha\ ior of 2mercaptobenzothiazole and 2-n1ei captobenzimidazole in aqueous buffer s o h tions and in nonaqueous media containing 0.5N sulfuric acid haq been described (151). Also, the polarographic behavior of thiourea and its derivatives has been used for analytical purposes (233). A conventional technique for the quantitative ectimation of thiol groups by reaction 11ith [1-(4-chloromercuriphenylazoj - 2 - ] naphthol has been investigated (588). The authors suggested modifications : incrcawd dilution of reactants. increased shaking, and lengthened time alloir c d fnr precipitation. A color test for thiols based on reaction n i t h .Y-ethvl maleimide in alkali n-as reported (%1. The determination of trichloroethane in dichloroethane n as made on the hasis of different stahility of the compounds to bases (149). A method for the detection and estimation of halogenated hydrocarbons is b a w l on the absorption of the hnlogenatcd hydrocarbons from the outer chani1m of a Conn-ay cell into the center cell, and the subsequent reaction with pyridine and alkali to form a pink color n-hich is measured spectrophotometrically (160). -4 colorimetric method used for the determination of oximes (~7%) is based on hydrolysis n ith acid and subsequent treatment n ith formaldehvrle and ferric alum. The extinction of the solution is measured at 515 mM. Hydroxylamine has been analyzed sgectrophotonietrically after reaction 1% ith 8-quinolinol to form 5&quinolinequinone-5(8-hydroxy-5-quinolylimide) (167). Dimethylaminobenzaldehyde in an aqueous alcoholic solution in the presence of acids has been adapted for the assay of hydrazine (174). In a Srrtndinai-ian
journal, a conventional technique for the determinabion of oxime nitrogen has been modified by the use of 2,4dinitrophenylhydrazine (554). The detection of unsubstituted para positions in phenols is based on oxidation with ammoniacal persulfate ions to colored products (473). Also, phenol groups may be determined conductometrically (432). A procedure has been described for determination of nz- and p-cresol in mixtures (269), based upon measuring the light absorption during color formation in reactions n-hich show extremes of light absorption. Several procedures h a r e been described for the determination and detection of catechol and pyrocatechol. The latter, a Colorimetric method, is based on the condensation of pyrocatechol and chlorosilene (28). Quantitative infrared d a h on catechol and resorcinol in solids using a fractional crystallization technique were described (648). Parsons, Seaman, and Woods (364) have described a spectrophotometric method for the determination of 1-naphthol in 2-naphbhol utilizing fractional precipitation followed by diazo coupling n i t h 2-naphthylamine-5,7-disulfonic acid a t a definite temperature and time. -4spot test for diketones and quinones, based on t'heir catalyt'ic effect in hastening the reaction between formaldehyde and 1,2-dinitrobenzene, has been reported by Feigl arid Xeto (157). Neiv microtests for anthracene. phenanthrene, and inositol \\ere made possible by conversion into anthraquinone, phenanthraquinonc. and cyclic: polyketones, respectively. Esters and anhydrides have h e n determined spectrophotometrically (282). The method is based on the formation of hydroxamic acid by interaction of esters or anhydrides, in alkaline solution with hydroxylamine. The hydroxaniic acids thus formed yield highly colored complexes with ferric ions. Benzyl benzoate and dibutyl phthalate were determined in mixtures by measurement of t'he absorbance a t 230 mp and by the quantity of standard alkali required to saponify the esters (116). The concentration of the esters was calculated by application of a differential equation. Phenol ethers \yere detected by the dark red color produced with sulfuric acid and formaldehyde (413). 4 quantitative method for p hydroxybfnzoic acid esters based on the ability of phenols to react Tvitli 4aminoantipyrene has been described (240). Acid-base titrations in ethylenediamine have been extended to include phenolic esters utilizing partition chromatography (180). Higuchi and Lachinan (215) demonstrated the retardation of the hydrolysis of benzocaine with caffeine; benzocaine T a s det,ermined by conventional spectrophotometry. VOL. 2 9 , NO. 4, APRIL 1957
707
”
Lindane has been determined colorimetrically in mushrooms by sulfonation of a methylene chloride extract (220). A method for the determination of Chlorbenside (p-chlorbenzyl p chlorophenyl sulfide) is based on oxidation of Chlorbenside to sulfone with hydrogen peroxide in glacial acetic acid, and nitration to the trinitro derivative, which develops a purple color with sodium methoxide in benzene (213). A precise and accurate method for 4-chlorc-2-methylphenoxyacetic acid by differential refractometry has been described (218). A method for the separation of pyrethrins I and I1 by circular chromatography was described (58). Szalkomski and Llader have described a spectrophotometric method for the determination of sodium carboxymethylcellulose in antibiotic preparations. The procedure is based on precipitation of the copper salt and colorimetric estimation with 2,7-dihydroxynaphthalene (@0). A nonaqueous titration technique has been applied to the assay of sodium carboxymethylcellulose (465). Preliminary heating with glacial acetic acid is necessary. A method of quantitative separation and recovery of certain quaternary ammonium bases by use of a Dowex-50 column was described ( I 7 2 ) . Simple paper chroniatographic techniques permit identification of the bases. The determination of quaternary ammonium compounds as phosphotungstates and colorimetrically as the bromophenol blue complex has been described (27, 688). A spot reaction for acidic polynitro compounds was described (155). Enolizable nitro compounds react with Rhodamine B to yield red-violet salts which fluoresce orange in benzene. The thermal decomposition of polyoxyethylene yields acetaldehyde, which forms a blue color with sodium nitroprusside and diethanolamine (414). Polyoxypropylene yields propionaldehyde, which produces orange colors. A colorimetric determination of polyethylene glycol mono-oleate depends upon the formation of a blue compound with (NH4)2[Co(C?rrS)4],which is extracted with chloroform (7’3). The absorption of the solution is measured a t 318.5 or 620 mp. A conventional technique for the determination of hydroxyl groups with propionic anhydride has been applied to polyethylene glycol (460). The determination of traces of benzene and toluene based on nitration and spectrophotometric measurements a t two selected wave lengths has been described (199). A colorimetric method for methoxyl groups is based on a conventional procedure for formaldehyde (316). The methoxyl group is hydrolytically cleaved to methanol and the latter is oxidized to formaldehyde. 708
ANALYTICAL CHEMISTRY
The formaldehyde thus obtained is determined colorimetrically after condensation with chromatropic acid. The development of new tetrazolium salts for determining reducing functions in organic compounds has been reported by Cheronis (93). At least two compounds were said to be seven to 10 times more sensitive than the wellknown triphenyltetrazolium chloride, when compared on the basis of their limits of detection of reducing sugars. Color reactions for flavone derivatives have been tabulated; hon.ever, most colorations are not suitable for use in colorimetric determination (360). For quantitative determination, the flavones reacted with hydrochloric acid and magnesium turnings in an ethanolic solution and absorption of the solution was measured a t approximately 500 to 550 mG, depending on the particular compound. A coulometric titration of dyes with externally generated titanous ion was reported by Parsons and Seaman (363). Titanous ion was generated electrolytically a t the mercury cathode using constant current, and has been applied to the titration of Orange 11, tartrazine, p-aminoazobenzene, amaranth, and methyl violet. The quantitative determination of ethylene glycol in water is based on periodate scission of glycols, follom-ed by formation of the insoluble silver iodate (212). The fact that 6 - ethoxy - 12 - dihydro - 2,2,4 - trimethylquinoline develops a strong fluormcence on exposure to ultraviolet light was used as the basis of an assay technique by Bickoff and associates (46). A differential spectrophotometric method for the determination of hexachlorophene in liquid soap has been described (95). The absorption of a solution a t p H 8 and a solution a t p H 3 was measured at 312 mp. A paper chromatographic separation was described for benzidine and its isomers (177). Best results were obtained with filter paper pretreated with formamide and TI ith cyclohexane as the solvent. Compounds were identified by color tests with p-dimethylaminobenzaldehyde or by diazotization follon ed by coupling TI ith N-l-naphthylethylenediamine. A colorimetric technique for the determination of azide ions is based on the formation of a red color mith iron(II1) (325). The color is presumably due t o Fen‘s and is stable below room temperature for 1 hour. Salts of organic bases may be precipitated with potassium bismuth iodide (KBiL) a t controlled p H values (78). The following bases were precipitated a t the p H given: caffeine, 1.2; codeine, 1.2; pentamethylenepyrrolidinium, 1.2; aminopyrine, 4; antipyrine, 1.5; and others. Aldehyde semicarbazones and ketone semicarbazides, when heated with di-
methylglyoxime and hydrochloric acid, give specific red to red-purple coloration (348). Thiosemicarbozones and thiosemicarbazides do not react, but the addition of 0- or p-hydroxybenzaldehyde and alkoxybenzaldehydes results in a red to red-purple coloration. Critchfield and associates (110) have described the determination of a,a-unsaturated compounds by reaction with morpholine in the presence of acetic acid as catalyst. The tertiary amine formed is determined by titration \T-ith acid after destruction of excess morpholine with acetic anhydride. Reproducibility is said to be within +0.2%. As little as 0.25 mg. of chloranil (tetrachlorop-benzoquinone) can be selectively identified with a 1% ethereal solution of tetramethyldiaminodiphenylmethane (166). The test may be used to detect other compounds that are converted into chloranil by heating the sample with potassium chlorate and concentrated hydrochloric acid. Methods for the detection of phenols, acyl derivatives of aromatic amines, arylurethanes, and monoarylureas, and sulfonic acid have been discussed by Feigl (152). Phenols are detected by their reaction 17-ith sodium cobaltinitrite in acetic acid and give a brown t o yellow color or precipitate. Acyl deriratives of aromatic amines, arylurethanes and monoarylureas, are detected by nitrosation followed by the addition of 1-naphthol; a red or orange color appears on warming. Sulfonic acids are detected by heating with alkaline sodium formate and ferriferricyanide to form Prussian blue. The determination of methoxyl groups in 1,2-dimethylpyrazdione and 1-phenyl-2-methylpyrazdione has been described (194). The paper chromatographic separation of n-alkyl sulfates, n-alkylpyridines, and m-alkyltrimethylammonium halides has been reported by Holness and Stone (219). BIBLIOGRAPHY
(1) Abbott, D. D., Rebes, L. A , , J . Am. Pharm. Assoc. 44, 287 (1955). (2)”,belson, D., Bondy, P. K., Arch. Biochem. and Biophys. 57, 208-17
(1955). (3) Achor, L. A., Geiling, E. M. K., AKAL.CHEY.26, 1061-62 (1954). (4) Blbers, R. IT., Lowry, 0. H., Zbzd., 27, 1829-31 (1955). (5) Allen, E., Seaman, W., Zbid., 27, 540-3 (1955). ( 6 ) Alt, L. L., Zbid., 27, 749 (1955). (7) Anastasi, A., Gallo, U., Novacic, L., J . Pharm. and Pharmacol. 7,
263-7 (1955). (8) Antoniades, H. N., Chemist Analyst 44, 78-84 (1955). (9) Ashton, G. C., Analyst 81, 228 (1956). (10) Ashton. C.. Brown, A. P., Zbid., 81, 220G.(1956). \
I
(11) Ashton, G. C., Foster, 11.C., Zbid., 80, 123-32 (1955).
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(1954). Knobloch, E., Chem. Listy 49, 26371 (1955). Knobloch, E., Svatek, E., Ibid., 49, 37-46 (1955). Kobrle, V., Zahradnik, R., Zbid., 48, 1703-5 (1954). Kolsek, J., Microchim. Acta 1956, 1193. Korpaczy, I., Zalta, I. P., Khouvine, J., Bull. S O C . chim. Biol. 35, 697 (1953). Kotarski, A., Blazkowska, Z., Roczniki Chem. 29, 849-61 (1955). Kovac, J., Chem. Zoesti 8, 342-5 (1954). Kovarik, hl., Vins, V., 2. anal. Chem. 147, 401-3 (1955). Krishnamurthv. K.. Swaminathan. M., ANAL. CHEM. 27, 1396 (1955): Krishnamurthy, K., Swaminathan, M., J . Sci. Ind. Research (India) 14, 310-1 (1955). Kritchevsky, D., McCandless, R. F. J., J . Am. Pharm. Assoc. 45, 385 (1956). Kryukov, P. A., Gidrokhim. Alaterialy 22, 90-5 (1954). Laitinen, H. A,, Woerner, D. E., ANAL.CHEM.27, 215 (1955). Lambert, J. L., Yasuda, S. K., Ibid., 27, 444 (1955). Lambiotte. hl.. Bull. SOC. chint. biol. 37, 1023-30 11955). Lane, E. S.. Analwst 80, 675-81 (1955). ' Laubie, H., Bull. SOC.pharm. Bordeaux 92, 135-6 (1951). Laughland, D. H., Phillips, W.Erf., ANAL.CHEM.28, 817 (1956). LeBlanc, K. F., Goddu, R. F., Wright, C. M . , Ibid., 27, 1251-5 (1955). Ledvina, hl., Chundela, B., Vecerek, B., Kacl, K., ceseslcoslov. Farmac. 4, 386-8 (1955). Leiserson, L., Walker, T. B., AKAL. CHEM.27, 1129-30 (1955). Levine, J., Fischbach, H., J. Am. Pharm. Assoc. 44, 543 (1955). Ibid., p. 713. Levine, U. E., Taterka, M., A w l . Chim. Acta 15,237 (1956). Lincoln, P. A., Chinnick, C. C. T., AnaZyst 81, 100-4 (1956). Litvenenko, L. M., Grekov, A, P., Zhur. Anal. Khim. 10., 164-8 ~.~ (1955). Loring, H. S.,Levy, I,. W.,Moss, L. K., AKAL. CHEX 28, 539 (1956). Lothe, J. J., Ibid., 28, 949 (1956). Lous, P., Acta Pharmacol. Toxicol. 6, 227 (1950). Lyness, W.I., Quackenbush, F. W., ANAL.CHEM.27, 1979 (1958). Lynn, W. S.,Jr., Steele, L. A , , Staple, E., Ibid., 28, 132 (1956). McAnally, J. S., Hausman, E. R., J. Lab. Clin. Med. 44, 647-50 (1954). hlcClure, J. H., Roder, T. hl., Kinsey, R. H., AKAL.CHEM.27, 1599-601 (1955). (297) Macek, K., Semonsky, M., Vanecek, S.,Zikan, V., Cerny, A , , Pharmazie 9, 752-4 (1954). (298) McKennis, H., Jr., Yard, A. S., ANAL. CHEM.26, 1960-3 (1954). (299) McKinley, J. D., Jr., Bull. Am. Soc. Hosp. P h a m . 12, 529 (1955). (300) McLafferty, F. W., ANAL.CHEM. 28, 3C6 (1956).
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(338) Neuwald, F., Ulex, G., Arch. Pharne. 288,432-8 (1955). (339) Ninni, L.,Ninni, M., Prakt. Akad. Athenon 29,452-65 (1954). (340) Niwa, H., Ann. Rept. Tdhoku Coll. Pharm. 1955,No. 2,9-12. (341) Noto, T., Matsuoka, G., Japan. Analyst 4,30-34 (1955). (342) Novotny, B., ceskoslov. Farmac. 4, 309-10 (1955). (343) Nowaczynski, W. J., Steyermark, P. R., Arch. Biochem. and Biophys. 58, 453-60 (1955). (344) Suller, G. D., Johnson, J. A,, hliller, B. S., A S A L . CHEM. 28, 884 (1956). (345) O'Connor, R. T.,J . Am. Oil Chemists' Soc. 33,1-15 (1956). (346)Ogawa, T., J . Chem. SOC. Japan 76, 739-44 (1955). (347) Ohkuma, S.,J . Pharm. SOC.Japan 75,1124-8 (1955). (348) Ibid., pp. 1249-52. (349)Zbid., pp. 1291-2. (350) Ohnesorge, W. E.,Rogers, L. B., ANAL.CHERL28,1017 (1956). (351) Okac, A., Jokl, V., ceskoslov. FarW Z ~ C 4, . 219-20 (1955). (352) Oreskes, I.,Saifer, A., ANAL.CHEM. 27,854 (1955). (353) Osol, A., Sideri, C. N., J . Am. Pharm. Assoc. 44,761-2(1956). (354) Owens, D. K., Sewage and Ind. Wastes 27,939-40(1955). (355) Owens, 1cI. L.,Mante, R. L., ASAL. CHEM.27,1177 (1956). Urbankova, J., Jehlicka, (356) Padr, Z., S., tkskosloa. Farmac. 4, 311-3 (1955). (357) . . Pan, S. C., AXAL.CHEM.27,65-7 (1955). (358)Pan, S.C., Dutcher, J. D., Ibid., 28, 836 (1956). (359) . , Pankratz. R. E., Bandelin, F. J.. J . Am: P h a k . Assoc. 45, 364 (1956). (360) Paris, R., Coruilleau, J.,Ann. pharm. franc. 13,192-9 (1955). Cox, R. J., Richards, D., (361) Parker, G., J . Pharm. and Pharmacol. 7,68391 (1955). (362) Parsons, J., Beher, W. T., ANAL. CHEW27,514-7 (1955). (363) Parsons, J. S.,Seaman, W., Zbid., 27,210 (1958). (364)Parsons, J. S., Seaman, W.. Woods, J. T.,'Zbid.,'27,21 (1955j. (365) Patton, J., Reeder, W., Ibid., 28, 1026 (1956). (366)Pechan, Z., Kalab, D., Palecek, E., Pharnzazie 10,526-8 (1955). (367) Pedley, E., J . Pharm. and PharmaCOZ.7,527-32 (1955). (368)Pekkarinen. A.. Pitkanen. M. E.. Scand. J : Clan. & Lab. Invest. 7; 1-7 (1955). (369) Perlin, A. S.,ANAL.CHEW27,396 (1955). (370) Pernarowski, RT., Drug Standards 23,157-S(1955). (371) Pesez, M., Ann. pharm. franc. 13, 513-6 (1955). (372) Peters, E. D.,Jungnickel, J. L., ANAL.CHEM.27,450 (1955). (373)Petrova. L. N.. Novikova. E. N.. Zhur.' Priklad. Khim. 28, 219 (1955). (374) Pillay, P. P., Rao, S, B., Rao, D. S., Indian J . Phamn. 17,95-7(1955). (375) Pinajian, J. J., Christian, J. E., J . Am. Phamn. Aseoc. 44. 631-6 (1955). '
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ANALYTICAL CHEMISTRY
(376) Platt, H., James, A. E., Zbid., 44, 666-8 (1955). (377) Poethke, W.,Horn, D., Pharnz. Zenlralhalle 94,41-5 (1955). (378) Poetke, W.,Trabert, H., Ibid., 91, 286 (1962). (379) Pohm, hl., Mikrochim. Acta 48, 1016-8 (1955). (380) Pohm, RT., Fuchs, L., Naturwissenschaften 41,63 (1954). (381) Polley, D., Muller, V. L., . ~ N A L . CHEW27,1162 (1955). (382) Pontius, D., Hoppe-Seyler's physiol. Chem. 298,268-71 (1954). (383) Pontius, D., Beckmann, I., Voigt, I(. D., Acta Endocrinol. 20,19-38 (1955). (384) Porter, C. C., ASAL CHEIZI. 27,805 (1955). (385) Pro, hf. J., Butler, W.P., blathers, A. P., J . Assoc. Oflc. Agr. Chemists 38,849-5'7(1955). (386) Puck, -4.. Klin. Wochschr.33,865-7 (1955). (387) Ramachandran, L. K., Epp, A., RlcConnell, IT. B., ANAL.CHEM. 27,1734-7 (1955). (388) Rausch, L.,Ritter, S., Klzn. Tt'ochschr. 33,1009-10 (1955). (389)Ravin, L.J., James, -4. E., J . A m . Pharm. Assoc. 44,215 (1955). (390) Reichelt, J., &skosloa. Fannac. 4, 297 (1955). (391)Reichelt, J., J . Pharna. and Pharmacol. 4,181 (1952). (392)Reid, \-. IT., Salmon, D. G., Analyst 80, 704 (1955). (393)Reifer, I.,Bull. zntern. acad. polon. sei. Classe 11, 3,207 (1955). (394)Reissig, J. L.,Strominger, J. L. Leloir, L. F., J . Biol. Chern. 217, 959 (1955). (395) Resnik, F.E.,Lee, L. A., Powell, W. A,. ASAL. CHERI. 27, 928 (1955): Stevenson, D., Samyn, (396) Ribeiro, D., J., Milosovich, G., Mattocks, A. Rl.. J . Am. Pharm. Assoc. 44. 226 (1955). (397) Rice, R. G., Kohn, E. J., h A L . CHEM. 27,1630 (1955). (398) Riemschneider, R., Keygand, C., Monatsh. 86, 201 (1955). (399) Robert, L.,Experientia 11, 316 (19553. (400) Rdbertson, D. M., Biochem. J . 61, 681 (1955). AXAL.CHERI.27, (401) Robinson, R. H., 1351 (1955). (402)Roche, J., Michel, R., Kunez, J , Bull. soc. chirn. biol. 37, 80'3 (1955). (403) Rodrigues, L.D., Silva, J. A., Rec. pori. farm. 5 , 75 (1965). (404) Rogers, A. R., Analyst 80, 903 (1955). (405) Rogers, W.,Jr., IGpnes, S. M., ANAL.CHEM. 27,1916 (1955). (406)Rohatgi, S.,J . Am. Pharm. Assoc. 44,428 (1955). (407) Romain, P., Clanet, F., Mesnard, P., Bull. soc. pharm. Bordeaux 92, 188 (1954). (408) Romain, P.,RIarzat, J., Mesnard, P., Ibid., 92,178 (1954). (409) Rondle, C. J. M.,Morgan, W.T. J.. Biochem. J . 61. 586 (1955). (410)Rose, H. h.,Ax.A'L. CHEM.26, 1245 (1954). (411)Zbid., 27,469 (1955). (412) Rose, H.A., J . Am. Pharm. Assoc. 44,414 (1955).
(413) Rosen, h4. J., AXAL.CHEM.27,111 (1955). (414)Zbid., p. 787. (415) Rosenblatt, D. H., Hlinka, Pa., Epstein, J., A N A L . CHEM. 27,1290 (1955). (416)Rosenblum, E. I.,Taylor, W.S., J . Pharm. and Pharmacol. 7, 1067 (1955). (417) Rosenkrantz, H.,Arch. Biochem. and Biophys. 44, 1 (1953); 46, 260 (1953). (418) Rosenkrantz, H.,Skogstrom, P., ANAL.CHERT. 28, 31 (1956). (419)Rotodaro, F. h., J . Assoc. Ofi. Agr. Chemists 38,809 (1955). (420) Rowley, K., Stoenner, R. W.,Gordon, L., A 4 ~C H~E h f~. 28, . 136 (1956). (421) Roy, A. C., Datta, S. X., Sur, R. N., J . Sei. Ind. Research (India)14, 124 (1955). (422) Rozsa, P., Magyar Kdm. Folydirat 61,122 (1955). (423) Ruch, J. E.,Johnson, J. B., ASAL. CHEM.28,69 (1956). Tousek, B., Hais, I. hl., (424) Rybar, D., Chem. Listu 48.1532 (1954). (425)Saifer, -4.,Oieskes, I., .&AL. CHEST. 28,501 (1956). (426) Sakaguchi, T . , Taguchi, K., Pharm. Bull. 3, 166 (1955). (427)Sakal. E.H.. llerrill. E. J.. J . Am. Phurm. Assoc. 43,709 (1954). (428) . . Sakurai. H..Chiba. S.. J . Pharm. Soc. j a p a h 75,106 (1955). (429) Saltzman, B. E.,ANAL.CHEM.27, 284 (1955). (430) Salvesen, B.,Domange, L., Guy, J., Ann. pharm.frang. 13,354(1955). (431) Sanchez, P Z.,Laboratorio (Granuda) 20,113(1955). 1432) Sarkanen. K.. Schuerch. C.. ANAL. CHEM. 27,1245 (i955j. ' (433) Sasakawa, Y.. J . Pharm. Soc. Japan ~. 75,946 (1955). (434) Savary, P., Desnuelle, P., Bull. SOC. chim. France 1954.936. (435) Schaffert, R. R., Kingsley, G. R., J . Biol. Chem. 212,59 (1955). (436) Schayer, R. W.,Kobayashi, Y., Smilev, R. L., Ibid., 212, 593 (1955). (437)Schenck, G.,Hannse, H., iirch. Pharwi. 287,544 (1954). (438) Schenck, G., Sadee, H., Ibid., 288, 101 (1955). (439) Scheurer, P. G.,Smith, F., ANAL. CHEM.27,1616 (1955). (440)Schilt, A. A, Smith, G. F., Heimbuch, il.,Ibid., 28,809 (1956). (441)Schmall, RI., Pifer, C. IT.,Wollish, E. G., Duschinsky, R., Garner, H., Ibid., 26,1521 (1954). (442)Schmidt, F., Gruhn, J.,AVaturwissenschaften 42. 391 (1955). (443)Schmi&t, F., Vorheier, D., Ibid., 42, 392 (1955). (444)Schmidt, H:, Staudinger, H., Biochem. 2. 326,343 (1955). 1445) Schmidt. 0..Manz. R., Klin. Wochschr. 33,857 (1955): (446) Schneider, F., Reinefeld, R., IIuller, H., Biochem. 2.327,189(1955). (447) Schneider, W . A . , Jr.,Streeter, L. E , h A L . CHEM. 27,1774 (1955). (448)Schoch, T. J., illaywald, E. C., Ibid., 28,382 (1956). (449)Schramm, M., Zbid., 28,963 (1956). (450) Schroeder, W., Voigt, K. D., Rec. trav. chim. 74.603 11955). (451) Schultz, 0.E ,' Strauss, D., Deut. Apoth. Ztg. 95,642 (1955). .
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Schweet, R. S., Biochim. et Biophys. Acta 18,566 (1955). Sciarra, J. J., Zapolosky, J. A., J. Am. Pharm. Assoc. 44,370(1955). Scott. Rf.. Taub. A , . Prantadosi., C.., Ibid., 45,232 11956). Scott, R. B., Schoeb, E. J., Vandenbelt, J. M.,Ibid., 44,377 (1955). Scott, R. W.,ANAL.CHEM.27,367 (1955). Scott, W.G., Taylor, R. T., Analyst 81,117 (1956). Secor, G. E.,White, L. Rf., ANAL. CHEM.27,1998 (1958). Serrano, J. F. V., Alves, A. C., Rev. port. farm. 5 , 67 (1955). Sezerat, A,, Ann. pharm. franC. 13, 516 (1955). Sharma. N. N., Anal. Chim. Acta 14,423 (i956j. Shell, J. W.,A N ~ LCHEM. . 27,1665 119.55). Shkr, J. 'H,, Ibid., 27,831 (1955). Shkodin, .4.hI., Tikhomirova, G. P., Ckrain. Khem. Zhur. 21, 265 (1955). (465) Siden, C. N., Osol, A,, J . Am. Pharm. Assoc. 44.759 (1955). (466) Siggia, S., Eichlin,' D. Lv., Rheinhart, R. C., ASAL. CHEY.27,1745 (1955). Siggia, S., Stahl, C. R., ANAL. CHEM.27,550 (1955). Zbid., p. 1975. Silberman, H., Thorp, R. H., J . Pharm. and Pharmacol. 5, 438 (1953). (470) Smith, D. C., Tompsett, S. L., dnalyst 80,397 (1955). (471) Smith. G. F..ANAL.CHEM.27. 1142 (1955). ' (472)Soboleva, 0. K., Aptechnoe Delo 4, 37 11RR5I. (473)SoionTay, L., Santoro, A., ANAL. CHERI. 27,798 (1955). (474)Sorensen, P., Ibid., 27.388(1955). (475)Souckova, hf., Zyka, J., c'eskosloz. Farmac. 4,181 (1955). (476)Spanyer, J. JF7., Phillips, J. P , ,%SAL. CHERf. 28. 253 (1956). (4'77)Stalder, G., Ann. Paediat. 176,270 (1951). (478) Stark, H.J., Klin. Vochschr. 34,153 (1956). (479) Stempel, B., Pharm. Zentralhalle 94, 263 (1955). (480) Stephens, J., Grainger, A,, J . Pharnz. and Pharmacal. 7. 702 (1955). Stock, F. G., Hinson, L. K., [bid. 7,512 (1955). Stoudt, T. H., Foster, J. K., Appl. Mzcrobzol. 2, 385 (1954). Streuli, C.A., h A L . CHEM.28,130 (1956). Siibramanian, S . ,Rao, M. V. L., J . Sci. Ind. Research (Indza) 14B, 566 (1955). Sudo, T., Shimoe, D., Miyahara, H., Japan. Analyst 4,88 (1955). Sullivan, M. X., Clarke, H. C. N., J . Assoc. Ofic. Agr. Chemists 38, 514 (1955). Svoboda, G. R., J . Am. Pharm. Assoc. 45,405 (1956). Swartz, C.,Foss, IC'. E., Zbid., 44, 217 (1955). Szalkowski, C. R., hlader, W. J., ANAL.CHEM.27,1404 (1955). Szalkowski, C. R.,Rlader, W. J., J . A m . Pharm. Assoc. 44, 533 (1955). (491) Szalkowski, C. R., RIader, W.J., J . Assoc. Ogic. Agr. Chemists 37,544 (1954). (492) Szalkowski, C. R., O'Brien, M. G., &fader, W. J., ANAL. CHEW 27, 945 (1955). \ - - - - ,
(493) Szalkowski, C. R., O'Brien, 31. G., Stewart, C. W.,hlader, W. J., Zbid., 38,140 (1955). (494) Takemoto, T., Daigo, K., Takai, T., J . Pharm. SOC.Japan 75, 1024 f 1955). Ibid., ~~'1025. Takemura, T., Science and Crime Detection 8,No. 3, 68 (1955). Tarpley, W.,Vitiello, C., $ p p l . Spectroscopy 9,69 (1955). Tauber, H., A N ~ L&Elf. . 27, 287 (1955). (499)Taufel, K., Pohloudek-Fabini, R., Z. Lebensm.-Untersuch u.-Forsch. 102,28 (1955). (500)Tepe, J. B., St. John, C. V., ANAL. CHERI. 27,744 (1955). (501) Theimer, E.E., Arnom, P., J . Am. Pharm. Assoc. 44,381 (1955). (502) Thies, H., Reuther, F. W.,'Vaturwissenschuften 42,462 (1955). (503)Zbid., p. 486. (504)Thies, H., Sorgenfrey, C. H., Reuther. F. IT., Ibid.. 42. 605 (1955). (505) Thomas, G., Roland, P.. Pharm. Weekb;lad90,129 (1955). (506)Thompson, S. S.,Analyst 81, 443 (1956). (507)Thoms, G. N.,Kotoinis, A. Z., Anal. Chim. Acta 14,457 (1966). (508) Tokar, G., Simonyi, L., Gal, G., Magyar IGm. Folydirat 61, 146 (1955). (509) Touchston, J . C., Hsii, C. T., h . 4 ~ . CHEM.27,1517 (1955). (510) Troll, W,,Lindsley, J., J . B ~ o l . Chem. 215,655 (1955). (511)Truitt, E.B., Jr., Morgan, A. RI., Little, J. M,, J . Am. Pharm. Assoc. 44,142 (1955). (512)Tschapke, H., Plessing, H., S a t u r wzssenschajten 42,417 (1955). (513) Tsiida, IC., lraruyama, LI., Ikek a m , S., J . Pharin. SOC.Japan 75,1309 (1955):. (514) Tsukamoto, T., Ijichi, S., Ibid., 75, 1016 (1965). (515)Tsunetomi. E.. Ibid.. 58.130 11955). (516jUdenfriend, S.;Tit&, E,', Ke;ksman, H., J . Biol. Chem. 216,499(1955). (517)Udovenko, V. V., Uvedenskaya, L. A,, Ukrain. Khim. Zhur.20,684 (1954). (518) Ultee, A . J., Jr., Hartel, J., ASAL. CHEM.27,557 (1955). (519) Underwood, A. L.,Ibid., 28, 41 (1956). (520)Underwood, J. C., Rockland, 1,. B., Zbid.. 26. 1553 (1954). (521) Urnberger,' E. J:, Ibid., 27, 768 (1955). (522) Vacek, J., eeskosloa. Farmac. 4, 6 (1955). (523)Valedinskaya, L. K.,Trudy ,$fed. Akad. 'Vauk S.S.S.R., dntibioiiki i ikh Primenenie 22. No. 1. 82 -(1952). Van Etten, C. H., sa^. CHEJI. 27, (524) 954 (1955). (525)Van Etten. C. H.. Earle. F. R.. McGuire; T. A.; Senti; F. R.: Ibid., 28,867 11956). (526) Vanllelle,. P. J., J . Am. Pharm. Assoc. 45,26 (1956). (527)Van Pinsteren, J. A. C., Verloop, & E.. I.Pharm. R'eekb!ad 90. 145 ~~(1955): Vinina. L. C.. Walsman. S. A,. Sci&e 120,389 (1954).' Vitte, G.,Guichard, C., Bull. SOC. vhurm. Bordeaux 93.31 (1955). Vogt, H., Arch. Pharm. 288, 20 (1955). I
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(531) Voldan, M., bskoslov. Farmac. 4, 407 (1955). Deut. Apoth.-Ztg. 1955, (532)Vollmer, K., 258. (533) Vonesch, E.E..Guagnini, 0. A., Anales asoc qulm. argentina 43,62 (1955). (534) Wacek. A.. Zeisler. F., Mikro. , Vonchim. Acta. 29 (1955): ' (535) . , Vulterin. J.. Zvka. J., Chem. Listu 48,1696 (1954).' ' (536) Waddington, D.J., Cullis, C. F., Anal. Chim. Acta 15, 158 (1956). (537) Wagner, H., Abisch, L., Bernhard, K., Helv. Chim. Acta 38, 1536 (1955). (538) Wagner, J., Kraus, P., Veverek, B., c'eskosloa. F a n a c . 4,389 (1955). (539)Walton, H.F., Smith, H. A., ANAL. CHEY.28,406 (1956). (540) Warner, B. R., Raptis, L. Z., Zbid., 27,1783 (1955). (541) . , bJ7arshowskv. B.. Schantz. E. J., Ibid., 26,"1811i1954). ' (542)Welsh. L.H.. J . Am. Pharm. .4ssoc. 44,507 (1955). (543)Wenger, P. E., Monnier, D., Faraggi, S., Anal. Chim. Acta 13, 293 (1955). (544)West, P. W,, Coll, H., ANAL.C H m . 27,1221 (1955). Sen, B., Ibid., 27,1480 (545) West, P. W., (1955). (546)Tf'estphal, O.,Feier, H., Luderitz, O., Fromme, I., Biochem. Z. 326, 139 (1954). (547) Whistler. R. I,.. Hickson. J. 1,. ASAL.'CHERI. 27, 1514 (1955). ' (548) Windrath, 0. C.. Ihid.., 28. . 263 (1956). (549)Wojahn, H.,Wempe, E., Arch. Pharm. 288,1 (1955). (550)Wolfgramm, C.,Weiss, F., Pharmazie 10,292 (1955). (551) Wollish, E. G., Colarusso, R. J., Pifer, C. W.,Schmall, M., A 4 s a ~ . CHEM.26,1753 (1954). (552) Woolford, 31. H., Jr., Chiccarelli, F. S., J . Am. Pharm. Assoc. 45, 400 (1956). (553) IJ.ootton, A. E.,Carruthers, -4,, Intern. Sugar J . 57,193 (1955). (554)Ynmada, T., Acta Chem. Scand. 9, 349 (1955). (555)Yamagishi, M., Xakamiira, I.., Sakajima, K., J . Pharm. Soc. Japan 74, 1354 (1954). (556)Yamaguchi, K., Fukushima, S., Tabata, T., Ito, M., Ibid. 74,1327 (1954). (557) Yamamoto, Y., Tomita, Y., Bull. Fac. .4gr., Kagoshima Unic. 3,114 (1954). Yemm, E. W.,Cocking, E. C., Analyst 80,209 (1955). Yoshino. T.. Sugihara. M . . Science and Ind. 29, 257 (1955).' Young, A., Sweet, T. R., Baker, B. B., ASAL. CHEM. 27, 356 (1955). (561)Zahradnik, R., Mansfield, V., Soucek, B., Pharnmie 10, 364 (1955). (562) Zbinovsky, U., XSAL. CHEY. 27, 764 (1955). (563)Zehner. J. M., Sweet. T. R.. Ihid.. 28,198 (1956). ' (564)Zimmer, A. J., Mansur, K., J . Am. Pharm. Assoc. 44,204 (1955). (565) Zinner, G.,Arch. Pharm. 288, 129 (1965). (566) Zyka, J., &skoslozJ. Farmac. 4,301 (1955). (567) Zyka, J., Pharmazie 10,170 (1955). .
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