(1977) Determination of Orthophosphate in Aqueous ...

Determination of Orthophosphate in Aqueous Solutions Containing Labile Organic and Inorganic Phosphorus Compounds1 W. A. Dick and M. A. Tabatabai 2 ABSTRACT A simple and precise colorimetric method of determining orthophosphate in aqueous solutions containing labile organic and inorganic P compounds is described. It involves a rapid formation of molybdenum blue color by the reaction of Orthophosphate with molybdate ions in the presence of ascorbic acid-trichloroacetic acid and citrate-arsenite reagents and complexation of the excess molybdate ions to prevent further formation of blue color from the phosphate derived from hydrolysis of the acid-labile P compounds. The color is stable up to 24 hours. The method is sensitive and accurate, and it permits determination of microgram quantities of Orthophosphate in samples containing large amounts of acid-labile P compounds. Tests with a wide range of condensed phosphate and organic phosphate compounds showed that none of the P compounds studied interfered with this method. Results by this method are compared with those obtained by the method of Murphy and Riley. Additional Index Words: water quality, phosphorus fertilizers.

Several methods have been proposed for determination of Orthophosphate in aqueous solutions. These include gravimetric, titrimetric, photometric, spectrophotometric, chromatographic, and radioactivation analysis (15). Some of these methods are tedious and time consuming, and others require special equipment and do not give reproducible results. Among the methods mentioned, however, the spectrophotometric heteropoly blue (molybdenum blue) color method is the most simple, sensitive, and accurate, especially for analysis of Orthophosphate at low concentrations. This method was developed by Osmond in 1887 (13), and since then, it has been modified by many workers. It involves the reaction of an acid ammonium molybdate solution with Orthophosphate ions to form a 'journal Pap. J-8518 of the Iowa Agric. & Home Econ. Exp. Stn., Arnes, Iowa. Projects 1868 and 2112. Received 11 June 1976. 2 Graduate Research Assistant and Associate Professor, respectively, Dep. of Agron., Iowa State Univ., Arnes, IA 50011.

82

J. Environ. Qua!., Vol. 6, no. 1, 1977

molybdophosphoric acid complex, which can be reduced to molybdenum blue in the aqueous phase (3, 7) or be reduced after extraction with an appropriate solution (4, 5, 8, 11, 14, 16). The most widely used procedure within the past decade, however, has been the one developed by Murphy and Riley (12). In this method, ascorbic acid is used as a reducing agent, and addition of antimony produces a molybdenum blue color in about 10 min that is stable for 24 hours. Several inorganic P(e.g.,Na tripolyphosphate, tetrasodium pyrophosphate, and Na hexaphosphate) and organic P compounds (e.g., adenosine triphosphate, fructose-6-phosphate, fructose-cliphosphate, and glucose-1-phosphate), however, interfere with this method. This interference may range from 2 to 10% for the condensed phosphates and from 0.1 to 10% for the organic P compounds present (6, 10), if the color measurement is made after 10 min. The percentage inference will increase with time of color development, because of hydrolysis of these P compounds in the acid medium. Pollution of water resources by phosphate derived from soils and fertilizers has generated interest in finding organic and inorganic P fertilizers that allow movement of P to the root zone and decrease phosphate fixation by soils, and thereby increase the fertilizer P use efficiency. Movement of such P compounds in soils allows their hydrolysis

by soil and root enzymes in areas where the fertilizer is needed for plant uptake. One of the major difficulties in studying these P fertilizers is associated with the methods used for determination of Orthophosphate in presence of the acid-labile organic and inorganic P compounds. Also, the organic and condensed P compounds are very widely distributed in nature. They are present in water (5), soils (C. P. Ghonsikar. 1970. Naturally-occurring polyphosphates in soils—Reactions of these and other polyphosphate materials in soils. Unpublished Ph.D. Thesis. Dep. of Agron., Ohio State Univ., Columbus, Ohio), and microorganisms (9). Their presence in aqueous solutions may

give misleading results of the amount of orthophosphate present in such samples. We required a simple, accurate, and specific method for determination of orthophosphate in the presence of various organic and inorganic P compounds. The method developed meets these requirements. It involves a rapid formation of molybdenum blue color from the reaction of orthophosphate with molybdate ions in the presence of ascorbic acid-trichloroacetic acid and citrate-arsenite reagents and complexation of the excess molybdate ions to prevent further formation of blue color from the phosphate derived from hydrolysis of the acid-labile P compounds. MATERIALS AND METHODS Reagents Ascorbic acid (0.1M)-trichloroacetic acid (0.5M) reagent agent A)-Dissolve 8.8 g of ascorbic acid and 41 g of trichloroacetic acid (Fisher certified reagent, Fisher Scientific Go., Chicago) in about 400 ml of water and adjust the volumeto 500 ml. This reagent should be prepareddaily. Ammoniummolybdate (0.01M) reagent (reagent B)-Dissolve 6.2 g of ammonium molybdate(J. T. Baker ChemicalCo., Phillipsburg, N.J.) in about 400 ml of water and adjust the volumeto 500 ml. Sodiumcitrate (0.1M)-sodiumarsenite (0.2M)-acetic acid reagent (reagent C)-Dissolve 29.4 g of sodium citrate and 26.0 of sodiumarsenite in about 800 ml of water, add 50 ml of glacial acetic acid (99.9%)and adjust the volumeto 1 liter. Standard phsophatestock solution-Dissolve 0.4390 g of potassium dihydrogen phosphate (KH2PO4)in about 700 ml of water and dilute to 1 liter with water. This solution contains 100/.tg of orthophosphate-P/ml. Water-Usedeionized water. Procedure Place 10 ml of reagent A in a 25-ml volumetric flask and add an aliquot (1-5 ml) of sample containing 2 to 25 /Jg of orthophosphate P, and immediatelyadd 2 ml of reagent B and 5 ml of reagent C. Swirl the flask to mix the contents after addition of the sample and each of reagents B and C, and adjust the volumewith water. After 10 min, measure the absorbance of the molybdenumblue color developed using a spectrophotometer adjusted to a wavelength of 700 nm. Calculate the orthophosphate P content of the aliquot analyzed by reference to a calibration graph plotted from the results obtainedwith standards containing0, 5, 10, 15, 20, and 25 //g of orthophosphate-P.To prepare this graph, pipette 0-, 5-, 10-, 15-, 20-, and 25-ml aliquots of the standard orthophosphate stock solution into 100-mlvolumetric flasks, makeup the volumes with water, and mix thoroughly, then analyze 1-ml aliquots of these diluted standards by the proceduredescribed for analysis of samples. In the work reported, the absorbance measurementswere obtained by using a BeckmanModelDB-Ggrating spectrophotometer with a 1-cmcell and a tungsten lampas the energy source. Equally good results were obtained by using a test-tube and a KlettSummerson photoelectric colorimeter fitted with a red (no. 66) filter. RESULTSAND DISCUSSION The method described is based on systematic studies of factors affecting formation of molybdenum blue color at room temperature and the removal of the excess molybdate ions by a citrate-arsenite reagent. The factors studied included concentration and amount of each of the reagents described. The method developed is similar to that

0.450

~1

~j 0.350 == 0.250 ~ "¢ 0.150

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0.050 400

500 800 700 800 900 WAVELENGTH (nm) Fig. 1-Absorbancespectra of molybdenum blue color produced by the methoddescribed: (1) 20/~gPO4-P;(2) 10/~gPO4-P; (3) 5/~gPO4-P. proposed by Baginski and Zak (i, 2) for determination phosphate and phospholipids in serum. Preliminary tests with the citrate-arsenite reagent (reagent C) indicated that, if properly used, this reagent complexes the free molybdate ions and stabilizes the molybdenum blue color complex produced from the reaction of orthophosphate with ammonium molybdate in the presence of ascorbic acid. Figure 1 shows the absorption spectra of the molybdenum blue color produced by the method described. Two absorption optima were observed; one at 700 nm and the other at 790 nm. These absorption peaks are different from those (720 and 882 nm) reported by Murphy and Riley (12). The first absorption peak is similar to that found by Beginski and Zak (1), but we could not detect the second peak (840 nm) that they reported. Beginski and Zak (1) used a Coleman Jr. spectrophotometer, while we used a Beckman DB-G spectrophotometer. It has been shown that when the molybdenum blue reduction takes place in about 13//tt2SO 4 or HC104, the highest peak at 840 nm is formed, whereas at lower acidities, a peak at 700 nm is formed (1). The linearity of changing the concentration of orthophosphate P was investigated and it was found that Beer’s Law was obeyed at 700 nm up to an absorbance value of 0.730 (30 t~g P). Murphy and Riley (12) reported that the molybdenum blue color produced by the reaction of orthophosphate and molybdenum in the presence of ascorbic acid and antimony reaches its maximumcolor intensity in both distilled water and sea water in about 10 min and remains constant for at least 24 hours. Tests indicated, however, that the time required for the molybdenumblue to reach maximumcolor intensity by the Murphy and Riley method depends on the amount of orthophosphate and composition of the aqueous samples under analysis. With some samples, between 20 and 30 min were required before a maximumintensity of the color was attained. All polyphosphates and some organic P compounds hydrolyze in the acidic medium recommended by Murphy and Riley (12). The molybdenum blue color produced from aqueous solutions containing such forms of P would not reach a maximumintensity until all the acid-labile P is hydrolyzed (the rate of hydrolysis of polyphosphate is a function of concentration of orthophosphate, acidity, and temperature of the medium). Figure 2 shows that the molybdenum blue color produced by the method described reached its maximumintensity after 10 rain. The results reported in Fig. 2 were obtained using a KlettJ. Environ.Qual., Vol. 6, no. 1, 197783

0.480

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0.400

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6’0 9~0 1:~0 10’O 0 REACTION TIME(minutes) Fig. 2-Effect of reaction time on the intensity of the molybdenum blue color produced by the methoddescribed:(1) 20/~gPO4-P; (2) 10/2gPO4-P;(3) 5 PO 4-P. Summersonphotoelectric coIorimeter fitted with a no. 66 filter and a test-tube. Similar results were obtained with a Beckman DB-G spectrophotometer. Tests showed that the intensity of the blue color produced would not increase in the presence of acid-labile inorganic phosphates, such as pyrophosphate or organic phosphates, if reagent C is added immediately after addition of reagent B to remove the excess molybdate ions. Delay in time of addition of reagent B and C or C, however, causes hydrolysis of the acid-labile phosphate and gives absorbance values higher than that expected from the orthophosphate present in the samples. Figure 3 shows results obtained when the addition of reagent B and C or C was delayed in times ranging from 10 to 60 min. Reagent C should be added immediately after the molybdate. Experiments in which either the citrate or arsenite was left out led to irregularities in color formation and instability of the color formed. Figure 4 shows the effects of 100 #g of P as pyrophosphate, trimetaphosphate, tripolyphosphate, and metaphosphate and time of molybdenum blue color measurement on the recovery of 5, 10, or 20/~g of orthophosphate P by the method of Murphy and Rile,/ (12). All polyphosphates tested interfered with the recovery of orthophosphate by this method, and the highest interference was found with the least stable polyphosphate; pyrophosphate. The degree of this interference increased as the amount of orthophosphate increased. To compare the results by the method developed, in the presence of organic and inorganic acid-labile P compounds, with those obtained by the Murphy and Riley method (12), analyzed aliquots (1-2 ml) of standard solutions containing 10 ~g of orthophosphate P or 10 /~g of orthophosphate P and 50/~g or 100 ~g of P as organic phosphate or polyphosphate. The absorbance of the blue color produced by each method was measured after 30 and 120 rain. The results obtained in this study are reported in Table 1, which indicates the response (as PO4~-) obtained from the different compounds with the two methods. None of the acid-labile phosphate compounds tested interfered with estimation of orthophosphate P by the method developed, but a-D-glucose-l-phosphate, phenolphthalein diphosphate, phosphoryl triamide, phosphonitrilic hexaamide, pyrophosphate, metaphosphate, trimetaphosphate, and tripolyphosphate increased the recovery of orthophosphate by the Murphy-Riley method. This interfer84

J. Environ. Qual.,Vol. 6, no. 1, 1977

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....... 20 30 40 50 60 REACTION TIME(minutes) Fi 9. 3-Effect of delay in time of addition of reagentB andC on the absorbanceof the molybdenum blu~ color producedfrom ~ of orthophosphate P in presenceof 500 ~ of pgrophosphate P by the methoddescribe~.Inset, effect of delay in time of addition of reauentC on recoveryof orthophosphate. " 0

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ence increased with increasing the amount of the acidlabile phosphate present in the aliquot analyzed. glycerophosphate and monomethyl phosphate did not interfere with the recovery of orthophosphate by either of the methods. Also, tests showed that the following P compounds did not interfere with analysis of orthophosphate by the proposed method: ~-naphthyl acid phosphate, triphosphopyridine nucleotide, D-glucose-6-phosphate, p-nitrophenyl phosphate, bis-p-nitrophenyl phosphate, phytic acid, and ammonium tetrametaphosphimate. Application of the ~nethod developed to analysis of orthophosphate P of soil extracts amended with pyrophosphate to contain 100 ~g P/ml showed that the pyrophosphate added did not interfere with orthophosphate analysis of the extracts tested (Table 2). 25

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3’0 6’0 9’0 1~ 0 REACTION TIME (minutes) Fig. 4-Effect of reaction time of color development on recoveryof various ~mounts of orthophosphate (OP)i~ the presenceof 100 ~ of P as pyropbosphate (PP), trimetaphosphate (TMP),tripolyphosphate(TPP), or metaphosphate{MP) by the method Murphyand ~iley (12): (A) 5~ PO4-P; (B) IO~PO4-P;(C) 20 ~ PO4-P.

Table 1—Recovery of 10 pg of orthophosphate P in the presence of some organic and inorganic P compounds by the MurphyRiley method (12) and the proposed method Orthophosphate P recoveredf Proposed Murphy Riley method method

Table 3—Precision of method Orthophosphate P analyzed

No. of analyses

Orthophosphate P recoveredf Range

« 1 5 10 15 20

Mean

SD

CV

0.03 0.08 0.05 0.07 0.27

3.1 1.6 0.5 0.5 1.4

r*6

6 7 8

0.95- 1.05 4.9 - 5.1 9.9 -10.1 14.9 -15.2 19.7 -20.2

0.98 5.0 10.0 15.1 19.9

A

B

A

B

0

10.0

10.0

10.0

10.0

a-D-glucose-1-phosphate

50 100

10.6 11.6

12.8 16.2

10.1 10.0

10.1 10.0

t SD, standard deviation; CV, coefficient of variation.

/3-Glycerophosphate

50 100

10.0 10.1

10.1 10.2

10.0 10.0

10.0 10.0

Monomethyl phosphate

50 100

10.0 9.9

10.0 9.9

10.0 10.1

10.0 10.1

Phenolphthalein diphosphate

50 100

10.8 11.4

11.1 17.0

9.9 10.0

9.9 10.1

Phosphoryl triamide

50 100

36.5 62.8

60.1 107.5

10.1 10.0

10.1 10.0

Phosphonitrilic hexaamide

50 100

11.9 14.7

13.2 18.2

10.0 10.0

10.0 10.0

Pyro phosphate

50 100

13.2 16.1

22.9 33.4

9.9 10.0

10.0 10.0

Metaphosphate

50 100

10.6 10.7

11.9 12.1

10.0 10.0

10.0 10.0

set of samples. If the intensity of the color produced from the aliquot analyzed exceeds that of 30 jug of PO4-P, take a smaller aliquot for analysis or use a 50-ml volumetric flask instead of the 25-ml flask recommended, add double the amounts of the reagents specified, and multiply the results by 2. Tests with a variety of cations and anions indicated that the recovery of 5 or 10 p.g PO4-P was quantitative (99.8veloped was made to contain 5 mg of K+, Na + , NH 4 + , Ca2+, or Mg 2+ , and NO 3 ~ or SO2,". The high precision of the method developed is illustrated by Table 3, which gives the results of replicate analyses of various amounts of PO4-P.

Trimetraphosphate

50 100

11.6 13.5

19.9 27.3

10.0 10.1

10.0 10.1

Tripolyphosphate

50 100

11.0 11.1

13.5 14.1

10.0 10.0

10.0 10.0

P compound None

P added

6 6

t Results calculated from absorbance measured after 30 min, A; and after 120 min, B.

Any colorimeter or spectrophotometer that permits color intensity measurements at 680-720 nm can be used for colorimetric analysis of the molybdenum blue color produced by the method described. The maximum absorption of the color measured is at 700 nm (Fig. 1), and the color is stable for at least 24 hours. With the exception of the results in Fig. 1, all results were obtained with a Klett-Summerson photoelectric colorimeter fitted with a red (no. 66) filter and a test tube. Standard curves prepared by the method described are highly reproducible; it is not necessary to prepare a standard curve with every Table 2—Recovery of orthophosphate in soil extracts by the method developed in the presence and absence of pyrophosphate (PP) Orthophosphate P Extractantf

Soil

+PPt

-PP

———— pg/mlBray no. 1

Webster Canisteo

2.5 1.4

2.5 1.3

Bray no. 2

Webster Canisteo

3.8 7.7

3.9 7.6

0.25AT HC1 + 0.25JV H2SO4

Webster Canisteo

6.4 19.8

6.2 19.8

W H2S04

Webster Canisteo

7.1 19.4

7.1 19.4

Water

Webster Canisteo

0.6 0.7

0.6 0.6

t Bray no. 1, 0.03JV NH 4 F + 0.025AT HC1; Bray no. 2, 0.037V NH 4 F + 0.1N HCL t The soil extract (3 g soil + 30 ml of extractant were shaken for 30 min and centrifuged at 12,000 rpm) was made to contain 100 ppm P as pyrophosphate (PP) before analysis for orthophosphate by the method developed.

J. Environ. Dual., Vol. 6, no. 1,1977

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