tion diminish the color yield, probably by occluding phosphomolybdate. Still, by inclusion of the appropriate controls, the method has been used for the study of such sluggish reactions as the direct hydrolysis of phosphocreatine in brain and muscle preparations. Most of the protein of such preparations is insoluble in the color development medium and can be centrifuged out easily. HA~O(-Zand sio3-z do not react in this method as as HP04-2: at equivalent concentrations HA SO^-^
gives 15% and SiOa-2 gives 10% as much color as HPOa-*, LITERATURE CITED
(1) Berenblum~ J V Chain, E., Biochem.
(2)J .Buell, 32, 286 M.(1938). v., Lowry, 0. H., Roberts, N. R., Chang, M. W , Kappahan, J. I., J . Biol. Chem. 233, 979 (1958). (3) Consden, R., Steiner, W.M., Nature 168, 298 (1951). (4) Dryer, R. L., Tammes, A. R., Routh, J. I., J . Biol. Chem. 225, 177 (1957).
(5) Fislte, C. H., Subbarow, Y., Zbid., 66,
375 (1925). (6) Lowry, 0. H., Lopez, J. A., Ibid., 162, 421 (1946). (7) Spector, L., Jones, M. E., Lipmann, F., in Colowick, S. P., Kaplan, N. O., “Methods in Enzymology,” Vol. 3, p. 653, Academic Press, New York, 1957. (8) Stadtman, E. R.,Iipmann, F., J. Biol. Chem. 185, 549 (1950). RECEIVED for review May 16, 1960. Accepted November 25, 1960. Work was supported by grant No. B-2048 from the National Institute of Neurological Diseases and BhdnesR.
Determination of Phosphorus in Biological Material W. T. OLIVER and
H. S. FUNNELL
Onfario Veterinary College, Guelph, Canada
b A method is described for the determination of elemental phosphorus in biological material. The tissue was mixed with tartaric acid in a flask fitted with an absorption train. The phosphorus was evolved b y heat in a nonoxidizing atmosphere and collected on mercuric bromide. It was eluted with iodine as phosphoric acid and the phosphorus determined b y the development of heteropoly blue. The method has been tested in a range from 0 to 54 pg. of phosphorus.
W
HITE PHOSPHORUS, though largely superseded by other agents as a pesticide, is still incorporated in some proprietary rodenticide formulations. In such forms, it occasionally causes accidental or malicious poisoning. Methods have been reported for the identification of this chemical in biological material (1-4, 6). The quantitative method presented here is based on the evolution of phosphorus from acid in a nonoxidizing atmosphere. Phosphorus is collected on mercuric bromide, then eluted from the absorption system as phosphoric acid, and determined colorimetrically as molybdenum blue.
EXPERIMENTAL
Apparatus and Reagents. The apparatus consists of a 125-ml. flatbottomed boiling flask equipped with a side arm and stopcock for the introduction of nitrogen. A Liebig condenser, total length 300 mm., is inserted into the neck of the flask and an absorption train composed of a scrubbing tube, a U-tube, and an absorption tube (5) is inserted into the top of the condenser. Standard-taper 24/40 joints are used in the flask, condenser, and bottom joint of the scrubbing tube. Standard-taper 12/30 joints are used in the remainder of the train. ,434
ANALYTICAL CHEMISTRY
Iodine solution, 0.02N. As described (5). Iodine solution, 0.003N. Dissolve 10 mg. of alginate (Dariloid K. B., Chas. Tennant Co., Toronto, Ont.) in 60 ml. of water by heating to 70” C. Cool to room temperature, add 15 ml. of 0.02~37iodine solution, and make up to 100 ml. with water. Prepare fresh before use. Ammonium molybdate solution. As described (6). Method. Prepare the absorption system by plugging the base of the scrubbing tube with cotton batting. Fill i t with fine silica sand; moisten the sand with 10% lead acetate solution and remove the excess solution with light suction. Place a small plug of batting in the absorption tube and by tapping the tube end gently, pack in 0.3 gram of mercuric bromide powder. Lubricate the joints with stopcock grease and connect the scrubbing tube and absorption tube by means of the U-tube. Clamp with elastic bands. Weigh 10 grams of finely divided sample into a 125-ml. modified boiling flask and connect the condenser to the flask. Close the stopcock and add 10 ml. of 1% tartaric acid down the condenser. Connect the rubber tubing from the nitrogen tank to the boiling flask by means of the side arm while opening the stopcock. Pass nitrogen through the system for 15 minutes and boil the contents for 45 minutes with continued gassing. Elution. Remove the absorption tube from the apparatus and insert it into the neck of a 10-ml. volumetric flask. Elute the powder with four 2ml. portions of 0.003N iodine solution, blowing the last few drops into the flask. Color Development. Add 1 ml. of molybdate solution and mix; add 0.5 ml. of freshly prepared 0.15% aqueous hydrazine sulfate solution, mix, and insert the flask in boiling water for exactly 10 minutes with
occasional shaking. Remove and cool rapidly in running water. Make up to 10 ml. with water, stopper, and mix by inversion. Read the color on a spectrophotometer a t 720 mp or a Klett-Summerson colorimeter using a No. 69 filter, in either case against a reagent blank. Compare the reading with a reference curve prepared from serial dilutions of a standard solution of monopotassium phosphate, reacted with the molybdate and hydrazine sulfate, made up to 10 ml. with distilled water and read against a blank of 8 ml. of distilled water carried through the procedure.
To determine the srnsitivity of the method, a standard solution of phosphorus wis prepared as follow: One 100-ml. and one 200-ml. volumetric flask were partially filled with chloroform, gassed with nitrogen for 5 minutes, and stoppered. A small piece of phosphorus (ea. 0.1 to 0.5 gram) was cut and washed by passing it successively through two beakers of distilled water and one beaker of absolute alcohol. It mas transferred from the ethyl alcohol wash into the 100-nil. volumetric flask and shaken mechanically until the phosphorus had dissolved. I t was made u p to volume with chloroform and 5 ml. were transferred to the 200-ml. flask and made up to volume with chloroform. An aliquot of this solution was added t o tissue in a previously gassed system and carried through the procedure. This solution was unstable and nas made up fresh for each determination. To assay the phosphorus solution, 1 ml. was added t o 8 nil. of freshly prepared, saturated chlorine water, shaken for 30 minutes, then heated on a steam bath to remove the chlorine. After cooling, 1 ml. of molybdate solution and 0.5 ml. of hydrazine sulfate solution were added and the flask mas heated for 10 minutes in a boiling water bath. I t was brought to room temperature
and the contents were made up to 10 ml. with water and read against a blank of 8 ml. of chlorine water and 1 ml. of chloroform carried through the procedure.
Table 1. Recovery of Known Amounts o f Phosphorus from Tissue
No. of Tests
Known amounts of standard solutions of phosphorus were added to 10 grams of macerated tissue and treated as described. The results are presented in Table I.
20 1 1
DISCUSSION
1
The method is rapid and reproducible. The instability of the standard solutions of phosphorus and the technical diffivulty of preparing standard solutions of precisely similar concentrations prerludcd replicate deteiminations of yield a t a particular concentration. HOTwer, within the range of phosphorus added (1 t o 54 pg.), the standard deviation in 16 trials between phosphorus added and rwovered (Table I) was 0.64 pg. with a standard error of mean differences of 0.16 pg. FollotTing evolution of phosphorus from tissue, iodine was used to elute the phosphorus from the absorption powder. Hoivever, iodine n-as found to be unsuitable as an eluent in the assay of the standard. The variable rmults in the latter case were inter~ ~ r e t easd due to the formation of varying amounts of the phosphorous acid.
1 1 1
1 1 1 1
I 1
Phosphorus, Mg. ReDisAdded covered crepancy 0 1.5 3.3 4.2 5.3 13.1 14.3 15.9 16.0 16.6 17.8 19.8 22.3 26.8 27.4 38.5 54.0
0 1.5 3.3 4.7 5.8 13.1 14.3 16.3 15.8 16.3 17.6 19.3 22.0 28.0 26.3 39.1 52.5
0 0 0 0.5 0.5 0 0 0.4 -0.2 -0.3 -0.2 -0.5
-0.3 1.2 -1.1 0.6 -1.5
As reported previously ( 5 ) , alginate was incorporated in the eluent to act as stabilizer for the colloidal molybdenum blue. The color is stable for a t least 1 hour and obeys Beer’s law within the range investigated here. Ninety micrograms of phosphorus was found to be the maximum concentration detectable with the amounts of reagents in this report. ACKNOWLEDGMENT
The authors gratefully acknowledge the technical assistance of Gerrie Smeenk. LITERATURE CITED
(1) Fresenius, C. R., “Manual of Qualitative Chemical Analyses,” p. 614, Wiles; New York, 1904. (2) Gonzales, T. A., Vance, M., Helpern, M., Umberger, C. J., “Lega;! Medicine,
Pathology and Toxicology, 2nd ed., Appleton-Century-Crofts, Kew York,
For this reason chlorine water was substituted eluent in the assay. The concentration of sulfide or niercapto groups in fresh tissue has not been found to interfere with recovery, and hydrogen sulfide added to the digestion flask as thioacetamide to a level of 50 pg. per gram of tissue did not affect recovery.
1954. (3) Kaye, S., J. Lab. Clin. Med. 28, 2 (1942). (4) Mitscherlich, E., Autenruth, W. v.,
“Laboratory Manual for the Detection
of Poisons and Powerful Drupe,” 5th ed., p. 5, Blakiston’s, Philadclphia, 1921. (5) Oliver, W. T., Funnell, H. S., ANAL. CHEM.31,259 (1959). (6) Scheuer, A,, Ann. Ckem. Phys. 112, 214 (1950).
RECEIVEDfor review May 16, 1980. Accepted December 5, 1980.
Spectrophotometric Determination of Va nadium(V) with N-Benzoyl-N-phenylhydroxylamine USHA PRlYADARSHlNl and S. G. TANDON Deparfrnenf o f Chemistry, Mahakoshal Mahavidyalaya, Jabalpur University, labalpur, India
b N-Benzoyl-N-phenylhydroxylamine has been found to b e a highly specific reagent for vanadium(V), forming with it a water-insoluble, deep violet complex in strongly acidic solutions. This violet complex, on extraction with chloroform, has been employed for the spectrophotometric determination of vanadium and for its separation from diverse ions.
uranium, manganese, aluminum, chromium, etc., which are conimonly associated with vanadium interfere with the direct determination of the metal and necessitate the use of masking agents or prior separation by ion exchange, electrodeposition, or precipitation. The precision and accuracy of the results are thus affected. Our work has revealed that N -
benzoyl-N-phenylhydrosylaminc,
A.
methods reported for the spectrophotometric determination of vanadium, the phosphotungstate ( I S ) , 8-quinolinol (g), benzohydrosamic acid (1, I I ) , and the recently discovered 3-tungstovanadic acid (10) methods are fairly sensitive and useful but not entirely adequate. The major defect of these methods is the lack of selectivity; one or more of the elements such as iron, cobalt, nickel, copper, thorium, VOKQ THE SEVERAL
O C ?-OH
=
O
forms a water-insoluble, violet complex with vanadate ions in strongly acidic solutions of 2 to 10M. This violet complex, on extraction with chloroform, forms the basis of a very sensitive and selective method for the spectrophotometric determination of minute
amounts of vanadium. Large amounts of Al(III), Co(II), Cr(III), Cu(II), Fe(III), hIn(II), Ni(II), Th(IV), U(VI), Zn(II), nitrate, and sulfate do not interfere n-ith the determination of vanadium. An attractive feature of the method is that it is relatively free from too many conditions. Many of the common variables such as acidity, ionic strength, temperature and volume of the aqueous phase, order of addition of reagents, amount of reagent used, etc., can vary under wide limits. The colored complex is formed instantancously and the stability of the system is adequate for analytical applications. The color develops completely in fairly strong acid solutions and this constitutes a distinct advantage because the complexes of many of the diverse ions with N-benzoyl-N-phenylhydroxylamine are decomposed in strongly acid solutions and, thus, vaVOL 33, NO. 3, MARCH 1961
435
