Spectrophotometric Determination of Phosphate in the Presence of

Charles M. Allen , Jr. and Mary Ellen Jones. Biochemistry 1964 3 (9), 1238- ... E. Raghupathy , N.A. Peterson , C.M. McKean. Biochemical Pharmacology ...
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Table I.

Per Cent Molybdenum in Five Analyzed Steels AV.

Runs Sample -1 B C

0 0 0 0 0

1)

1 405 215 206 093

0 0 0 0 0

2 396 223 200 100 060

Values, 3 0 396 0 218 0 192 0 096 0.065

% 0 0 0 0 0

399 219 199 096

E 067 064 Metallurgj- Laboratory, Electric Auto-Lite Co., Toledo, Ohio.

slon-ly through the resin (2 to 3 ml. per minute). When the level of the initial aliquot is just above the top of the resin, wash down the wdls with deionized water, and adjust the flow rate to 8 to 10 ml. per minute until the total volume collected is slightly less than 300 ml. Adjust to p H 2 with 4N sodium hydrazide. Finally dilute the effluent to 300 ml. with deionized water which has been passed through the column. Mix the solution thoroughly and let stand for several minutes for full color development. Transfer a portion of the red-orange complex to a sample tube and place in the colorimeter previously set a t zero absorbance with water at a ware length of 550 mp, the wave length of maximum absorbanre for the complex. RESULTS A N D DISCUSSION

At a wave length of 550 mp, absorbances for the standard samples containing 0.100, 0.200, 0.350, and 0.500%

Certified Values,"

% 0 0 0 0 0

41 22 20 090 07

molybdenum were 0.132, 0.220, 0.350, and 0.490, respectively. The standard low-alloy steel also contained l.OOyo chromium, 0.80yo manganese, and O.lti7' nickel. The absorbance readings of the standards correspond to transmittance values of from 73.5 to 32.3%, which are very close to the optimum operating range for the Spectronic 20. The results obtained from the analysis of five l o r alloy steels are shown in Table I. These values were obtained from a t least triplicate samples in every case. Duplicate aliquots were run for each of the triplicate samples. The phenylfluoronemolybdate complex was stable a t p H 2 for 24 hours but tended to show some decomposition after standing for longer periods of time a t room temperature. During the development of the procedure, it was found that molybdate ion TI as not completely removed from

the coluinn. Hoiwver, by adding a small amount of citric acid t o the solution as suggested by Klenient ( y ) , molybdate ions \wre rffectirely removed from the resin. From the results obtained by Luke and Campbell (4), LT-ho ran tests on 59 metals to determine which ones yielded stable colored phenylfluorone chelates, it \\-as concluded that our procedure gave an effluent free froin interfering ions. S o further studies on inberierences \!--ere niade. LITERATURE CITED

( I ) Goldstein, G., Manning, I>. C., llenis, O., h x . 4 ~C. H m f . 30, 539 (1958). ( 2 ) Kolthoff, I. N.,Sandell: E. B., "Textbook of Quantitative Inorganic Analysis," 3rd ed., p. 692. Macniillan, Sew York, 1952. (3) Luke, C. L., A X A L . CEEM.28, 1276 (1956). (4)Luke, C. L., Campbell, 11,E., Ibid., 28, 1273 (1956). ( 5 ) Otterson, D. h.,Graah. J. If-., Ibid., 30, 1282 (1958). ( 6 ) Pecsok, R. L .,--Parkhurst. It. AI., Ibid., 27, 1920 (193s). ( i )Samuelson, O., "Ion Exchangers in -4nalytical Chemistry," p. 153. Kiley, Sew Tork, 1953. (8) Katerbury, G. R., Bricker. C. E., AYAL.CHEM.29, 129 (1957). (9) Kill, Fritz, 111, Poe, J. H.. Ibid., 2 5 , 1363 (1953).

RECEIVED for review January S. 1Bti0. .\ecepted December 8, 1960. Division of .\nalytical Chemistry, 135th Meeting, .1CS, Boston, AIass., Ipril 1959.

Spectrophotometric Determination of Phosphate in the Presence of Highly Labile Phosphorus Compound LEWIS C.

MOKRASCH'

Neurochemistry Laboratory, Section on Experimental Neurology, Deparfmenf of Medicine, University o f Kansas Medical Center, Kansas City 72, Kan.

b By substituting N,N-dimethylformamide for most of the water in the phosphate assay, the hydrolysis of labile phosphorus compounds is virtually arrested and b y reading the color a t 335 mp, a much more sensitive method i s obtained. The molar absorptivity for phosphate in this procedure i s 17,500. Applications and permissible variations in procedure and sources of interference are discussed.

N

uhmxows colorimetric methods for phosphate are based on the reduction of phosphomolybdate, of which three differ in some essential respect (1, 5 , 6). These methods have certain defects which limit their universal ap432

ANALYTICAL CHEMISTRY

plication, such as too high acidity, sensitivity to sulfhydryl compounds, or the need for extractions. I n a n attempt to find a phosphate assay milder than the Lowry-Lopez method and more sensitive, the possibility of combining the molybdenum blue color with benzidine blue in an acidic alcoholic medium was investigated. Although no blue color appeared immediately, the strong yellow color found in the samples containing phosphate was much more intense than the normal color of phosphomolybdate. Efforts to improve the reliability resulted in the evolution of a simple method which is suitable for the determination of phosphate in the presence of labile phosphorus compounds. ;iscor-

bic acid is the reducing agent, replacing benzidine, and acetic acid-acetate is the buffer system in a diniethylformamide medium. MATERIALS

K,HPOI was recrystallized and dried 2 hours a t 110' C. for use as the phosphate standard. A-,S-Dimethylforniamide (DAIF) vias redistilled through a 1S-em. Widmer column a t about 50% reflux and the fraction boiling a t l53O + 0.5' was collected. Conimercial diniethylformamide may give aatis1 Present address, Research Laboratory, AlcLean Hospital, Belmont, RIass.

factory results \I ithout redistillation if the compound does not smell strongly of dimethylamine. Carbamoyl phosphate n a s prepared by the method of dpector et al. ( 7 ) and acetyl phosphate by the method of d t d t m a n and Lipinanii (8). METHOD

n

< 3233-

The color reagent i q coinposed of 1 volume of 8.1in.11 (SH4)Jlo7Os4HsO, 5 volumes of 0.54.lf acetic acid-0.05M potassium acetatc-0.03m-11 CuS04 and 20 volumes of diinethylfornianiide. Although customarily made shortly before use, the shelf lift. of the mixture is more than a day. On longer standing a white precipitate appears. .4 2.50-ml. amount of the color reagent is inixed n i t h a 0.50-ml. sample containing 0.1 pmole or less of phosphate, and the thoroughly mixed solution is allowed to stand a t room temperature. At a convenient time. 2 to 10 minutes after mixing and the same for all samples. 0.10 mi. of 0.05731 ascorbic acid is added to each tube with immediate mixing. The color may be read after 5 riiinutcs a t 335 m p . Samples nhich are turbid, such as those containing protein, may be centrifuged during the color development period. This I\ ill likely btl necessary when the s:impli~is a complete reaction mixture for an enzyme acsay.

5

C

IO

I5 20 NIn Utes

25

3C

Figure 1 . Rate of color development for phosphorus compounds Curve 1. 1.0 pmole ocetyl phosphate plus 0.1 2 1 pmole H P 0 4 - 2 Curve 2. 0.1 02 pmole H P 0 4 - * Curve 3. 0.50 wmole carbamoyl phosphate plus 0.079 pmole H P 0 4 - 2 Curve 4. 1 .O pmole phosphocreatine plus 0.035 pmole HPO4-2 Curve 5. 1.0 pmole adenosine triphosphate Curve 6. 0.5 pmole carbamoyl phosphate Curve 7. 1 .O pmole phosphocreatine Curve 8. 1 .O pmole acetyl phosphate

phosphate. The low acidity and replacement of most of the water by D I I F result in a marked stabilization of the labile phosphorus compounds. Needless to say, the normally stable phosphorus compounds are inert in this system and are not reported here because this method deals with the problems originating in the use of labile conipounds. The color rise for the labile phosphorus compounds follows zero order kinetics between 10 and 30 minutes or more. This permits easy estimation of contaminating phosphate by extrapolation of the color development curve to the ordinate and makes it unnecessary to calculate the pseudo first-order kinetic constants as is necessary for accuracy when the Lowry-Lopez method is used. Table I indicates the variations in technique which may be introduced for the sake of convenience without sub-

DISCUSSION

The amount of color developed varies somewhat, depending in part upon the lrngth of the timr the test solution is allowed to stand with tlie color reagent bc.fore the introduction of ascorbic acid. With a phosphate standard, the c d o r yield ordinarily increases about 7% as this period increases from 1 to 10 minutes. For a 10-minute period the maximum molar absorptivity is 17,500. The inclusion of standards with each analysis is recommended. The absorption maximum of 335 mp agrees with the results of Consden and Steiner ( 3 ) . When benzidinr is used in place of ascorbic arid, the high absorption of benzidine itself in this region makes the procedures less precise. 4Aininodiphenylamine (A' - phenyl - p phenylenediamine), which is a good reducing agent in more acid media ( 4 ) .is not active enough in the acetate buffer chosen to be useful in this method. The relative rates of color development for phosphate and other compounds after the addition of ascorbic acid to the mixtures are illustrated in Figure 1. I n the absence of other compounds, the color development for phosphate is virtually complete in 2 minutes. I n the presence of the compounds, it is somewhat retarded, but is still complete a t 10 minutes in all cases. The other phosphorus compounds have no effect on the amount of color given by added

*

3 33-

Table

I.

Permissible Variations Procedure

Variable Amount of buffer Cu +* concentration I

Acetic acid concentration Dimethylformamide Ascorbic acid Ammonium molybdate Time before addition of ascorbic acid Color development

in

Rangea f50 70 -507, to

f100-fold

3~507~ -207, to 10% &50% &50yc 1 to 10 minutes

+

2 to 30 minutes

a Ranges in terms of percentage refer to the normal quantities described in the text.

stantially altcriiig tlie siicitir it? 01 belectivity of the nietliod. The order of addition of reagents to the pliospliatc sample as stated in JIethocis is obhgatory. If the aicorbatc is addcd to the color reagent prior to it3 addition to the phosphate solution or if the mol\ bdate is addcd last, the color !.icld nil1 bc greatly diminished. Substitution of mothanol. etliniiol, or 2-propanol for D l I F results in :I lonex color yield, a higher rate of tlecbonipositioii of phosphocreatine and a higher salt sensitivity. Substitution oi diiiiethylsulfoxide for D l I F inhibit> the plioqphomolybdate ieduction. Similarly, increaqing the amount of DJIF in the system inhibits color development and decreasing the amount of DMF acceleratcs the decomposition of labile compounds. There ia great profit in avoiding the use of peichloric acid or trichloroacetic acid to stop an cnzymic assay. The DIIF ;tops the rcaction and stabilizes thc labile compounds. Interferences. Adenosine tiiphosphate (ATP) gives an anomalous reaction in this system. .\pparciitly it and C U +conibine ~ to form a yellon complex, the color of nhich fades slonly. For a given amount of -ITP, the color is roughly proportional t o tlie amount of Cu f Z in the color reagent. The interference by salt is rouglily proportional to the qalt concentration. K i t h an eltraneous salt (Wac1 or 2hydroxymrthyl - 1.2 - dihydroxypropane-2-ammonium chloride) concentration of 5 m X in the completctl reaction mixture, there is a 10ycdecrease in color. This effect is decreased n hcii the DAIF concentration is decreased and increased sharply n hen the D Y F concentration is increased over t h a t given in Methods. Obviously, the salt effect is minimized by avoiding the use of high ionic strengths in the media t o be analyzed, n hieh is easily accomplished in all but very few cases. The interference by -SH compounds can be partly reversed. Cysteine, 1.3 m J I (final reaction mixture), decreases the color by 927,, but the addition of S-ethylmaleimide, n hich binds -SH groups, to 3mJf results in a net decrease of color of only 21%. The usc of Sethylmaleimide is not necessary n here interference due to -SH groups is sufficiently controlled by the C U * ~in the reagent used as described, and this is the case most of the time. Thus, C U + ~ has been used satisfactorily in the analysis of glycogen phosphorylase, n hich requires -SH compounds as cofactors. The addition of more Cu"? is similarly effective in binding -SH groups but if the test solution contains rlTP, the high ATP blank ~$111be encountered. Protein is a poqsible source of interference. High protein concentrations (more than 2 mg. ml.-I) in the tebt soluVOL. 33, NO. 3, MARCH 1 9 6 1

433

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). for review May 16, 1960. AcRECEIVED cepted 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. I n 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 a r m and stopcock for the introduction of nitrogen. A Liebig condenser, total length 300 mm., is inserted into the neck of the flask and a n 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 n a s 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