Table 111.
Determination of Nitrogen (Stannous Chloride Added)
Compound 1,2,4,6-Tetramethylpyridiniumiodide
1,2-Dimethylpyridinium iodide 3-Carbamido-1-methylpyridiniumiodide 1-Methylpyridinium iodide Phenyltrimethylammonium chloride
Theory 5.32 5.94 10.62 6.34 5.32
Percentage of Nitrogen Av. Standard Found deviation deviation 5.23 10.01 0.009 5.84 10.03 0.040 10.57 & O . 03 0,038 6.29 10.03 0.033 5.26 f0.03 0.028
tin-foil sample cup is convenient and the resulting tin salts have a beneficial effect on the reaction. ACKNOWLEDGMENT
The authors are grateful to E. M. Kosower for preparation and purification of the pyridinium salts. LITERATURE CITED
sample cup and stannous chloride solution, equivalent to about 140 mg. of tin, was added prior to digestion. Results are compatible with the original nitrogen values. Measurement of the digestion mixture, using a calibrated iron-constantan thermocouple, showed a temperature of 335’ C., which remained essentially constant, regardless of the presence or absence of tin. A plausible explanation for the difference in nitrogen values obtained in the presence of tin would be the catalytic effect of tin salts in the removal of iodine. Hot concentrated sulfuric acid, an oxidized agent, readily oxidizes iodide to elemental iodine. Any hydriodic acid formed is converted to free iodine. As the reaction proceeds, the iodine formed is gradually distilled out of the digestion flask. However, as the iodine is formed in the digestion mixture, it may react indirectly 1%-ithammoniacal nitrogen being produced, converting it to free nitrogen (10). It is postulated that iodine, and other halogens, may be oxidized to oxyhalogen
acids, which in turn oxidize ammonium compounds (10). The results of these experiments indicate that the presence of tin salts in the digestion mixture prevents the formation of oxyiodine acids. The loss of nitrogen attributed to the action of iodine or its compounds is usually small and may be induced by addition of iodide after the sample has been digested. Thus the reactions involved concern the ammonium salts and/or the mercury-ammonia complex formed when mercury is present in the catalyst. These reactions appear to be absent in the presence of tin salts. The chloride salt was not affected by the presence or absence of tin. For the chloride ion, the reaction of sulfuric acid forms hydrogen chloride, which is readily vaporized from the digestion media. CONCLUSION
The semimicro-Kjeldahl method gives excellent recovery of nitrogen from a wide variety of pyridinium halide and oxyhalide salts (4, 6). The use of the
Bradstreet, R. B., AXAL.CHEM.26, 185 (1954). ,Bradstreet, R. B., Chem. Rets. 27, 331 (1940). Clark, E. P., “Semimicro Quantitative Organic Analysis,” Academic Press, Xew York, 1943. Cole, J. O., Parks, C. R., IND.ENG. CHEST.,API‘AL.ED. 18. 61 (1946). Crane. F. E..Fu O S S . R. M., ANAL. CHEW26, 1651 (1954). ’ Fish, V. B., Ibid., 24, 760 (1952). Friedrich, A., Kuhaas, E., Schnurch, R., 2. physiol. Chem. 216, 68 (1933). Gautier, J. A., Renault, J., Ann. chim. anal. 28. 85 (1946). Marzado, M.,’ Mskrochemie ver. Mikrochim. $eta 36/37,671(1951); 38,372 (1952). Modeer, E., Univ. Wyoming Pub. 7,NO.2,13-26 (1940). Ogg, C. L., Brand, R. W.,Tillits, C. O., J . Assoc. O j i c . Agr. Chemists 31, 663 (1948). Shirley, R. L., Becker, W. W., IXD. EXG. CHEM., ANM.. ED. 17, 437 (1945). Willits, C. O.,Coe, 11.R., Ogg, C. L., J . Assoc. Ofic.Agr. Chemists 32, 118 (1949). RECEIVEDfor revieF March 28, 1957. Accepted August 19,1957.
Determination of Phosphorus in Organic Compounds Rapid Micro and Semimicromethod KENNETH D. FLEISCHER, BURNETT C. SOUTHWORTH, JOHN H. HODECKER, and MURRAY M. TUCKERMAN Sterling- Winthrop Research Institute, Rensselaer, N. Y.
b An organophosphorus compound may b e burned in an oxygen-filled fiask by the Schoniger method. All phosphorus is converted to the orthophosphate, which may b e determined either titrimetrically or colorimetrically. The colorimetric method is based on the formation of heteropoly blue and is used for a microdetermination. The phosphate may also b e precipitated as magnesium ammonium phosphate hexahydrate. The precipitate is then dissolved in acid and the amount of magnesium determined with (ethylenedinitri1o)tetraaceticacid. The micromethod is accurate to within 270, 152
ANALYTICAL CHEMISTRY
while the semimicromethod is accurate to within 0.5%.
I
rapid method for the microdetermination of halogens and sulfur in organic compounds (6, 7 ) , place the weighed sample in a filter paper envelope, affix to the end of a platinum wire, and burn in an oxygen-filled flask. Absorb the desired element in a suitable liquid absorbent on the bottom of the flask and then titrate. The ease, rapidity, and accuracy of this method recommend that it be extended to other elements. Schoniger ( 5 ) predicted its N A
application to the determination of phosphorus, arsenic, and metals. This paper describes the combustion of organophosphorus compounds by the above procedure followed by either of two methods for the determination of the phosphorus. For semimicroanalysis the authors employed a modification of the method of Flaschka and Holasek ( 2 ) . Precipitate the phosphate as magnesium ammonium phosphate hexahydrate. The salt concentration present affects this precipitation and therefore the lower limit in this case appears to be about 1 mg. of phosphorus. Collect the precipitate on a frit and wash thor-
oughly. Then dissolve it in acid, add an excess of standardized (ethylenedinitri1o)tetraacetic acid, and adjust the pH to beta-een 8 and 10. Back-titrate the excess (ethylenedinitrilo) tetraacetic acid with a standardized solution of magnesium chloride, using Eriochrome Black T as the indicator. Several attempts were made to avoid this back-titration, but erratic results were obtained. Apparently, the mechanics of this precipitation are sensitive to the conditions of the glassware, stirring, salt concentration and other factors. If the approximate amount of phosphorus present is known, add a portion of the complexone required before the solution is made ammoniacal, thus avoiding a back-titration. The microprocedure is based on the colorimetric measurement of the heteropoly blue formed by the phosphate as compared to a standard (4). APPARATUS A N D REAGENTS
The only special apparatus needed is the platinum wire sealed in a 24/40 ground-glass stopper. The mire should have a spiral perpendicular to it, or a net, to hold the sample about 1 inch above the bottom of a 300-ml. Erlenmeyer flask. TThatman paper, No. 54, was used for the combustions. Most of the reagents used were common laboratory items. Standardized 0.01S solutions of magnesium chloride and (ethylenedinitri1o)tetraacetic acid are required. A 0.1% mixture of Eriochrome Black T in sodium chloride was employed as an indicator. bIngnesia mixture. Dissolve 50 grams of magnesium chloride hexahydrate and 100 grams of ammonium chloride in 1 liter of mater. Colorimetric reagent. Mix equal volumes of 2.5y0 (mt./vol.) ammonium molybdate in water and 1% (wt./vol.) bismuth subcarbonate, U.S.P., in 7N sulfuric acid. Immediately before use, add 10 grams of ascorbic acid per 100 ml. of the above solution. The low pH of this solution is required for proper color development. Standard phosphate solution. Dissolve 0.1098 gram of reagent grade potassium dihydrogen phosphate in 100 ml. of nater. Dilute a 2-ml. aliquot of this solution to 250 ml. with water. There are 2 figrams of phosphorus per milliliter of solution. PROCEDURE FOR SEMIMICROANALYSIS
Weigh a sample containing about 1 mg. of phosphorus on a tared piece of filter paper 1 inch square, folded into an envelope. Add a fuse of filter paper and affix the whole to the platinum wire. Introduce 5 ml. of (I to 2) nitric acid into a 300-nil. Erlenmeyer flask and fill the flask with oxygen. Ignite the fuse and plunge the stopper with the sample attached into the flask. When combustion has ceased, shake the flask vigorously so that all the phosphorus pentoxide is taken up in the
Table I. Accuracy of Method
Error, Partds/lOOO P Found, % Calcd., yo Micro Semimicro Micro Semimicro - 17 0 6-Allyl-6,7-dihydro-5H-dibenz9.29 9.16 9.29 9.33 (c,e)azepinephosphate (Ilidar f 4 Phosphate") 9 30 +I 0 9.29 -4 9.25 +9 -4 3-Diethylamino-2,2-dimethylpro- 7.64 7.73 7.61 propyl tropate phosphate (Syntropan Phosphate") +10 -12 3,4Dichlorobenzyl-triphenyl6.77 6.84 6.86 phosphonium chloride -14 +29 Ethylenediamine salt of mono11.73 11.59 12.08 guaiacylphosphoricacid a Registered trade marks Hoffman-LaRoche, Tutley, N. J. Name
acid. Heat the solution to the boiling point momentarily t o assure the complete conversion of phosphorus to orthophosphate. Transfer the solution to a 100-ml. beaker with a minimum amount of distilled water. Add 2 ml. of the magnesia mixture and one drop of a suitable indicator. Make the solution ammoniacal and add a few drops in excess. The precipitate forms slowly and becomes visible in 1 to 5 minutes. Allow it to stand in the cold for 2 to 4 hours. A longer time results in a coarser precipitate, but the recovery is the same. Filter the precipitate through a medium porosity frit, then wash thoroughly with 5% (wt./vol.) ammonium hydroxide. The volume of filtrate and washings is about 125 ml. Dissolve the precipitate in 1 ml. of 1N hydrochloric acid and transfer it to a 100-ml. beaker. Add exactly 10.00 ml. of 0.01.V (ethylenedinitrilo) tetraacetic acid and follow with 2 ml. of 3N ammonium hydroxide. Add about 100 mg. of the indicator mixture and titrate the solution with 0.01X magnesium chloride to a red color. Deduct an indicator blank of 0.03 ml. of solution from the volume of magnesium chloride used. PROCEDURE FOR MICROANALYSIS
Burn a sample containing 0.2 mg. of phosphorus as before and boil the solution. Then transfer it to a 100-ml. volumetric flask. Use a 2-ml. aliquot for the determination. I n test tubes graduated a t 10 nil., introduce 1, 2, and 3 ml. of the standard phosphate solution and aliquots of solutions to be analyzed. Dilute each one to 7 ml. Add 2 ml. of the colorimetric reagent, dilute to exactly 10 ml., mix the contents, and allow the solutions to stand for 5 minutes. Read the transmittance in a colorimeter using a filter having a maximum transmittance a t 660 mfi. Compare the unknown solutions m-ith the standard. DISCUSSION
Hillebrand and associates (3) state
that there is a small loss of phosphorus into the filtrate and washings during the precipitation of magnesium ammonium phosphate. An examination of the combined filtrate and washings, using the colorimetric method described above, demonstrated that there is a loss of 0.025 mg. of phosphorus. This is negligible compared with the total phosphorus in a macrodetermination, but this amount must be included in the calculations for the semimicromethod, The reagent blank, determined colorimetrically, amounts to 0.00038 mg. of phosphorus and may be neglected. The precipitate need stand only 2 to 4 hours before filtration. Other errors inherent in the precipitation have been studied ( 3 ) . Other elements forming heteropoly acids with molybdates which are reducible to molybdenum blue are silicon, arsenic, and germanium. Silicon, as silicate, does not interfere with the colorimetric method, Arsenic may be separated from the phosphate and determined quantitatively by the method of Jean, but arsenic occurs rarely with phosphorus in organic compounds. Germanium is infrequently encountered in organic analysis. That the combustion is accurate and precise has been demonstrated by Schoniger (6, 7 ) . The procedure is used routinely now in this laboratory for the determinations of halogens, sulfur, and phosphorus. Table I indicates the accuracy and applicability of the method. The first two samples were received as standard samples for a collaborative study of phosphorus analyses under the auspices of the iissociation of Official Agricultural Chemists. The other tm-o samples were research compounds prepared a t the Sterling-Kinthrop Research Institute and may be considered to be a t least 9770 pure based on elemental analysis for carbon, hydrogen, and nitrogen or chlorine. Applying the method of Dean and Dixon ( 1 ) to the semimicromethod, the VOL. 30, NO. 1, JANUARY 1958
153
standard deviation, su, is 0.03%. From the range, the 95% confidence interval is =!=0.03% and the 99% confidence interval is 10.05%. CONCLUSION
The method developed by Schoniger for the combustion of organic compounds has proved most satisfactory for the determination of several elements, and might be applied t o several more. Its extreme simplicity, as compared to the conventional Pregl combustion and other common methods, has actually hampered its growth in some areas. The above results indicate that phosphorus in organic compounds may be deter-
mined rapidly and accurately by this method.
RECEIVKDfor review April 12, 1957. Accepted October 16, 1957.
LITERATURE CITED
Interferenceswith Biuret Methods for Serum Proteins-Correction
( 1 ) Dean, R. B., Dixon, W. J., ANAL. CHEY.23,636 (1951). (2) Flaschka, H., Holasek, A., Mikrochemie ver. Mikrochim. Acta 39, 101 (1952). (3) Hil!ebrand, W. F., Lundell, G. E. F.,
Bright, H. A., Hoffman, J. I., (‘Applied Inorganic Analysis,” 2nd ed., p. 702, Wiley, New York, 1953. (4) Jean, hI., Anal. Chim. Acta 14, 17282 (1956). (5) Schoniger, W., Midatlantic Analytical
Symposium, Philadelphia, Pa., Feb. I ^ _ _
8 , 1w/.
(6) Schoniger, W., 1955, 123. (7) Zbid., 1956,869,
iMikrochim. Acta
In the article on “Interferences with Biuret Nethods for Serum Proteins, Use of Benedict’s Qualitative Glucose Reagent as a Biuret Reagent” [ANAL, CHEU. 29, 1491 (1957)], in the first column on page 1492, the first line of the directions for preparation of Benedict’s qualitative glucose reagent should read 173 grams of sodium citrate. RICHARD J. HESRY CHARLESSOBEL SAMBERKMAN
165. Yohimbine Hydrochloride GORDON BURLEY and GEORGE M. BROWN National Bureau of Standards, Washington 25, D. C., and University of Maryland, College Park, Md.
unit cell dimensions, space group, and optical refractive indices of yohimbine hydrochloride (C21H280,N2. HC1) have been determined. Cell constants were obtained from rotation and precession photographs and the parameters were refined from a powder diffraction pattern. Yohimbine hydrochloride was recrystallized from a commercial preparation by Mallinckrodt. Organic microTHE
analysis (in per cent) gave C = 64.67, H = 7.16, and N = 7.37, compared to the calculated values of C = 64.51, H = 6.98, and N = 7.17. Microscopic examination of the crystals showed them t o be very thin plates, usually triangular in outline, but also often rhombic or six-sided. The indices of refraction were measured using the sodium D line (589 mN). The symmetry is orthorhombic.
‘7/ ’
CHaOOC-
I
OH Structural formula for yohimbine hydrochloride
X-RAYCRYSTALLOGRAPHIC DATA X-Ray Power Diffraction Data for Yohimbine Hy‘drochloride 2 6 0 ~ .2ecaied. d hkl 1/11 d hkl 1 / 1 1 r)eobed. 28caled. 7.07 7.07 12.475 020 v8 28.10 28.02 3.182 O i l wm 8.39 8.39 10.040 110 vw 28.50 28.52 3.127 080 m 29.34 29.33 3.041 270 wm 13,12 13.07 6.763 130 30.05 30.05 2.970 212 vw 13.14 6.732 011 31.21 31.20 2.864 052 trace 14.16 4 15 6 249 040 ms 35.72 35.73 2.510 450 VW 15.26 5 26 5 801 200 m 38.14 38.14 2.358 0 10 1 m 16.11 6 09 5 500 140 wm 39.09 352 16.49 6 53 5 355 031 wm 39.02 38.95 2.307 1 1 0 1 wm 18.23 8 23 4 860 131 m 38.92 510 18.70 8 62 4 760 230 vw 19.32 19.30 4.595 150 m 11.77 41.77 2.160 203 vw 19.91 19.87 4 462 201 m 42.57 42.63 2.119 2 11 0 vw 20.24 20.19 4 392 211 w 292 20.55 20.52 4 324 141 w 41.36 44.42 2.038 1 0 2 m 21.30 21.29 4.168 060 8 45.24 45.20 2.004 1 vw 22.65 22.64 3 924 160 m 46.07 46.08 1.968 462 vw 23.17 23.15 3 837 151 w 49.05 49.12 1.853 353 vw 24.14 24.07 3 693 320 w 3.419 161 26.03 3.416 170 26.62 26.65 3.341 102 vw 51.12 51.11 1 . i 8 5 093 w
if: $
154
ANALYTICAL CHEMISTRY
Unit Cell Dimensions. a = 11.59 i.0.02 A. b = 24.97 & 0.10 A. c = 6.99 j=0.02 A. Formula Weights per Cell. 4. Density. 1.284 grams per cc. (27.24’ C.) by volume displacement, 1.282 grams per cc. by x-ray. Axial Ratio. a : b : c = 0.4642:l: 0.2709.
Space Group. D23- P21212.
OPTICALCRYSTALLOGRAPHIC DATA = 1.57, p = Refractive Indices 1.61, y = 1.69. Optic Axial Plane. 010 dcute Bisectrix. y = a CRYSTALLOGRAPHIC DATA for publication in this section should be sent to W. C. NcCrone, 500 East 33rd St., Chicago 16,
111. Submitted by Gordon Burley in partial fulfillment of the requirements for the M.S.degree, Chemistry Department, Cnivereity of Maryland, 1952.
