Table
I.
Estimation of Known Amount of Atropine
Taken, mg. 0.20 0.72 0.98 1.30 1.73
Atropine Calcd., mg. 0.20 0.70 0.96 1.27 1.5
Relative error, yo nil 2 2 2 13
very small, and thus it was impractical to calculate the result. Similarly, for amounts higher than 1.3 mg. the results are erroneous. The amount of total alkaloids determined in Datura alba roots and leaves was 0.188 and 0.027%, respectively. One of the main advantages of this method over the colorimetric method is
that no calibration with the pure sample is required. Besides, this method can be used for Datura alba crude extracts whereas for colorimetric estimation the crude extract should be complet,ely free from impurities and coloring matter. The method is still simpler in chlorophyll-free parts of plants, where pretreatment with chloroform is not required, and the powdered material is simply processed for the extraction of alkaloid. While estimating a known amount of atropine, alcohol was tried as a percolating medium instead of chloroform for the elution of alkaloid from the column, but the results obtained were not accurate. ACKNOWLEDGMENT
The authors are indebted to S. A. Warsi, Director, North Regional Laboratories, P.C.S.I.R., Peshswar, for his help in preparing the paper.
LITERATURE CITED
(1) Davidek, J., Blattn6, J., J . Chromafog. 7,204 (1962). ((2) Freeman, F. M., Analyst 80, 520-2 (1955). (1955). (3) General Medical Council, “British Pharmacopoeia,” p. 75-6, Pharmaceutical Press, London, 1958. (4) Martin, E..W., Cook, E. F., “Remingtons Practice of Pharmacy, 11th Ed., p. 242, Mack Publishing Co., Ertston, Pa., Pa 1956. 1Qm (5) M&icBk, M&ic N., Mikrochim. Acta. 2, 283-8 (1961); Anal. dbstr. 8, 389 f1961). (6)’ Mukerji B., “The Indian Pharmaceutical dodex,” Vol. I, p. 37, Council of Scientific & Industrial Research, New Delhi 1953. ( 7 ) 0801, A., harrar, G. E., “The Dispensatory of United States of America,” 25th Ed., p. 142, Lippincott, Philadelphia, 1955. (8) Ro&yriska,M., Goszyriska, K., Farm. Polsku 8, 421-2 (1952); C.A. 47, 9561e (1953). (9) Worrell, L., Booth, R. E., J . Am. Phurm. Assoc. 42,361-4 (1953). RECEIVED for review February 11, 1963. hccepted May 14, 1963.
A New Digestion Reagent for the Determination of Microgram Quantities of Phosphorus in Organic Compounds PAUL
M. SALIMAN
Shell Development Co., Modesto, Calif.
b A new digestion reagent containing hydriodic acid, calcium iodide, water, phenol, and acetic acid was developed for the determination of phosphorus in organic compounds to the nearest 0.2 pg. During removal of solvent and excess reagent by volatilization and combustion, phosphorus is converted to orthophosphate. The molybdenum blue color is developed in the original container. The procedure can b e adapted to either ultramicro or trace analysis, and is applicable to organic phosphorus in a wide variety of solvents. Also, a rapid semimicro procedure is described which utilizes digestion in sulfuric and perchloric acids, followed b y formation of the phosphovanadomolybdate complex.
T
amount of research on phosphorus compounds, especially in their biological applications, has required the development of suitable analytical techniques, and much work has been done along these lines as evidenced by the wealth of articles ap. pearing in the literature. These procedures vary from combustion in an oxygen flask ( I , 2) to persulfate (6) or hydrogen peroxide oxidation (4). The HE EVER-INCREASING
112
ANALYTICAL CHEMISTRY
use of nitric acid (6) or perchloric acid (3) for digesting plant materials is not uncommon. All these procedures are designed to convert phosphorus to orthophosphate. The molybdenum blue and other colorimetric methods for determination of phosphorus in organic compounds are normally dependent upon the quantitative conversion of phosphorus to orthophosphate. The type of sample and expected concentration determines the procedure to be used for conversion to orthophosphate. For instance, the oxygen flask cannot be used for compounds in aqueous solution or for nonaqueous solutions containing less than 100 p.p.m, of phosphorus because of sample size limitations. Wet digestions, using strong oxidizing reagents, are sometimes difficult to apply to solutions of relatively volatile phosphorus compounds such as 2,2-dichlorovinyl dimethyl phosphate or O,O,S-trimethyl dithiophosphate or to solvents which are relatively nonvolatile such as glycol or grease. The use of strong oxidizing reagents with some solvents may even be hazardous. Because of the limitations of strong oxidizing procedures and because of the solvents, concentrations, and com-
pounds encountered in biological investigations, a reagent and a method of wider application were needed. In biological investigations, problems in the determination of phosphorus vary from analysis of a few micrograms of relatively pure material to analysis of phosphorus in aqueous or nonaqueous solutions containing as little as 10 p.p.b. phosphorus. These low concentrations are often encountered when the investigation involves initial isolation by gas liquid, liquid-liquid, or paper chromatography, or separations by extraction. For maximum utility, a procedure should be applicable to aqueous or nonaqueous solutions of a wide variety of materials and cover a wide range of phosphorus content. The reagent and procedure described below appear to satisfy these requirements. EXPERIMENTAL
Reagents. Prepare phosphorus-free HI by adding 25 grams of iodine to 125 ml. of 477, H I containing 1.5y0 HsPOz as preservative. Distill, collecting the first 115 to 125 ml. of distillate. The distilled H I will be colored with iodine. The reagent is prepared by adding 50 ml. of t,he distilled HI, 0.6 gram
Table I. Analyses of Typical Compounds, S'emimicro Sample (pure compounds) (KH~POIStandard) Trimethyl phosphite
PhoSPhoruSi mg: Added Found
1.95 1.92 3.16 2.83 P,P-dibutyl-N,N2.08 diisopropylphosphinic 1 . 1 6 amide 0.70 Triphenyl phosphine 0.92 2.22 1 .oo 1.92 Methyl parathion 1.83 1.29 Phosdrin 2.88 2.22 insecticide Phosdrin insecticide, 2.99 1 % on Pyrax dust 2.62 1-(Dichlorophosphinyl) 2.90
1.89 1.90 3.13 2.82 2.10 1.18 0.72 0.93 2.22 0.99 1.93
5 empty gelatin cap-
0
0
0
0
piperidine
sules, size 5 Silicic acid, silica gel, 1 gram
1.85 1.31 2.86 2.19 2.99 2.49 2.87
is outlined below. The sample in a 100-ml. volumetric flask is decomposed completely in less than 5 minutes by heating on a hotplate with 3 ml. of sulfuric acid plus 0.5 ml. of 70% perchloric acid. Samples containing more than 75 mg. of organic matter may require pretreatment before addition of perchloric acid, such as boiling off solvent, charring sample in the sulfuric acid, or incremental addition of perchloric acid to the hot solution. After the solution cools, water, ammonium vanadate, and ammonium molybdate solutions are added and the absorbance determined at a calibrated wavelength between 400 mp and 470 mp. While the sulfuric acid and perchloric acid have been described by others, the overall procedure has been simplified so that it requires less than 30 minutes and is easily applied to the analysis of many samples per day on a routine basis. All organic phosphorus compounds so far tested have yielded their phosphorus to this digestion (Table I). A very few volatile compounds required a hydrolysis period a t room temperature in the stoppered flask, or the addition of bromine water to ensure complete recovery of phosphorus. DISCUSSION A N D RESULTS
Ca(OH),, 50 ml. of water, and 500 grams of phenol to a 1-liter flask. The solution is made up to 1 liter with acetic acid. Ammonium molybdate solution, 2.5 grams of (NH~)BMO&'~H~O per liter of 1.25N H2S04. Hydrazine sulfate solution, 0.6 gram of (NHz)zH2S04per liter of water. Procedure. T o the samples preferably containing les3 than 8 p g . of phosphorus in a 30- or 50-ml. crucible or beaker, is added 2 ml. of reagent. The covered crucible is placed on a .team bath and the solution is digested for a period of time which depends upon the phosphorus compound under study. The cover is removed and solvents and excess reagent removed by volatilization. The crucible is transferred to a hot plate to speed the process. For safety and to increase the speed of removal, c o r bustible solvents may be ignited into open flame. Removal of all carbonaceous residue is completed by combusting the residue in a furnace a t about 700' C. for 5 to 10 minutes. After cooling, 4 ml. of ammonium molybdal e solution and 1 ml. of hydrazine sulfate solution are added. Full color ir; developed by heating the covered criicible on a steam bath for 10 minutes. Absorbance is determined a t a wayrelength of 830 m p . Since 8 p g . of phlmphorus produce an absorbance of about 1.3 using the volumes above, a sample containing more than 8 p g . of phosphorus requires dilution with the color-forming solutions or the use of a thmner cell. Semimicro Procedure. A rapid, easily performed and reliable method for determining from 40 pg. to 5 mg. of phosphorus in organic compounds
Determination of lo\v-molecularweight alkoxy1 groups of phosphate esters by use of aqueous HI in phenol was applicable to all insecticides tested; a'HI-containing reagent, as described
above, was therefore tested as a means for recovering phosphorus. Using this reagent, phosphorus was successfully determined in such compounds as 2,2-dichlorovinyl dimethyl phosphate, trimethyl phosphate, methyl parathion, O,O,O-tri-p-tolyl phosphorothioate, 0,0,O-triethyl phosphorothioate, tricyclohexyl phosphine oxide, Phosdrin insecticide, and others. I n addition to being applicable to these materials as an ultramicro method for phosphorus determination, the method was applicable as a trace method for phosphorus in the order of a part per million or less in various media, such as organic matter, water, lubricating oil, carbon tetrachloride, acetone, acetic acid, xylene, glycol, and mineral oil. Recovery of phosphorus from compounds dissolved in methanol, ethanoi, and isopropanol, all of which might be expected to react with the HI, was also quantitative. Apparently the reaction of H I with the phosphate is much more rapid than with the alcohol solvent. Table It. Recovery of Orthophosphate after Ignition a t 700' C. in the Presence of an Excess of Three Cations 3.7 pg. of P added as HsPO, Recovery, pg. Ignition time, min. 10 20 Ca 3.6 3.8 Ka 3.0 0.9
I
