OF PHOSPHORUS. IN RIVER WATER BASED ON THE REACTION OF VANADO-. MOLYBDOPHOSPHATE. WITH MALACHITE GREEN. SHOJI MOTOMIZU* ...
Analytica Chimica Acta, 211 (1988) 119-127 Elsevier Science Publishers B.V., Amsterdam -
119 Printed in The Netherlands
SPECTROPHOTOMETRIC DETERMINATION OF PHOSPHORUS IN RIVER WATER BASED ON THE REACTION OF VANADOMOLYBDOPHOSPHATE WITH MALACHITE GREEN
SHOJI MOTOMIZU*, MITSUKO OSHIMA and ATSUSHI HIRASHIMA Department of Chemistry, Faculty of Science, Okayama University, Tsushimanaka, Okayama 700 (Japan) (Received 13th January 1988)
SUMMARY The formation of an ion-associate between vanadomolybdophosphate and malachite green in aqueous acidic solution (0.5 M sulfuric acid) enables trace amounts (O-l x 10e5M) of phosphate to be determined. The molar absorptivity is 1.05 X lo5 1 mol-’ cm-’ at 620 nm. The complex was stabilized in solution by adding poly (vinyl alcohol). Other ions generally found in river waters did not interfere. Interference by silicate is less than that found in the corresponding malachite green molybdophosphate procedure; interference of arsenate is avoided by reduction with thiosulfate. The method is applied to the determination of ,ng 1-i amounts of phosphorus in river water; the results obtained were in good agreement with those obtained by an extraction-spectrophotometric method with malachite green.
Most procedures for the spectrophotometric determination of phosphorus are based on the formation of heteropolyacids such as molybdophosphate and vanadomolybdophosphate in acidic medium. Molybdophosphoric acid and its reduced product, phosphomolybdenum blue, are used as a light-absorbing species either as they are or after extraction into an organic solvent as a protonated species as an ion associate with a bulky cation. Motomizu et al. [ 1,2] have reported sensitive extraction-spectrophotometric methods for the determination of phosphate with molybdate and cationic dyes: the molar absorptivities with ethyl violet and malachite green were 2.7 x lo5 and 2.3 x lo5 1mol-’ cm-‘, respectively. Although the methods are very sensitive and are hardly affected by co-existing ions, they are troublesome in routine work, because of the necessity for extraction. Itaya and Ui [ 31 reported a simple and sensitive spectrophotometric method for the determination of phosphate in serum with molybdate and malachite green; the method was subsequently modified and applied to the determination of phosphate in serum, plasma and urine [4-111. The mechanism of the coloration reaction between molybdate and malachite
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green has been studied by Altmann et al. [ 121. Fogg et al. [ 131 used crystal violet instead of malachite green but the method required heating of the solution. Motomiiu et al. [ 141 improved the method reported by Itaya and Ui, and applied it to the determination of phosphorus at sub-mg 1-l levels in river water; the improved method was subsequently applied to the determination of phosphorus at pg 1-l levels in river water by the flow injection method [ 151. Recently, TBei et al. [ 161 reported the use of guinea green B instead of malachite green; the method was more sensitive than that with malachite green, but time-consuming. The vanadomolybdophosphoric acid method has been widely used for the determination of phosphorus [ 17,181; this ternary heteropoly acid is said to be more stable and absorbs more strongly near 400 nm than the molybdophosphate but the sensitivity provided is less than that obtained with phosphomolybdenum. The reaction of a ternary heteropoly acid with a cationic dye has been little studied, though it seems that its coloration is more selective for phosphate than silicate and arsenate. In this work, the color reaction of vanadomolybdphosphate with malachite green and its application to the spectrophotometric determination of phosphorus in river water were studied. EXPERIMENTAL
Apparatus and reagents
Absorption measurements were made with a Hitachi Model 139 spectrophotometer and a Shimadzu Model UV 300 recording spectrophotometer in quartz cells of lo-mm path length. All chemicals used, except malachite green and poly (vinyl alcohol), were of analytical-reagent grade. The malachite green solution (5 x 10m3 M) was prepared from the commercially available malachite green oxalate in distilled water or in diluted sulfuric acid (0.005 M). The ammonium molybdate solution (0.5 M) was prepared from (NH,), (Mo702.,) l 4H20 (Nakarai Chemicals) in lop4 M sulfuric acid. The lo-’ M solution of ammonium vanadate (Wako Pure Chemical Industry) was prepared in distilled water. Standard phosphate solution. Potassium dihydrogen-phosphate was dried at reduced pressure (about 5 mm Hg ) at 60’ C to constant mass. The dried compound (0.2722 g ) was dissolved in distilled water to give 11 of solution (2 x 10v3 M); it was diluted accurately to give working solutions. Working solutions should be prepared fresh daily, and should be acidified with sulfuric acid to 0.005 M. PolyCvinyl alcohol) (PVA) solution (5% w/v). Commercially available PVA
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217 and 405 (number average degree of polymerization 1700 and 500, respectively) was dissolved in hot water. Non-ionic surfactants examined were Brij-35 [ CH,,H,,O ( CH&H20)23H], Brij-58 [ C16HS30 ( CH&H,O)zoH] ,Emulgen-120 [ C,,H,,O (CH,CH,O) IS.7 H], Emulgen-950 [n-CSH,S*CGH,0(CHzCH20)50H] and Triton X-100 [ C,H,,*C,H,O (CH,CH,O) l0H]. These were dissolved in distilled water. Preparation of reagent solutions. Reagent solutions were prepared for each purpose by mixing molybdate, vanadate and diluted sulfuric acid solutions as follows. Reagent solution A (for determining orthophosphate in water ) was prepared by mixing 40 ml of 0.5 M molybdate solution, 8.3 ml of lo-’ M vanadate solution, 22.8 ml of concentrated sulfuric acid and 28.9 ml of distilled water. Reagent solution B (for determining orthophosphate and total phosphate in river waters) was prepared by mixing 60 ml of 0.5 M molybdate solution, 12.5 ml of low2 M vanadate solution, 22.0 ml of concentrated sulfuric acid and 5.5 ml of distilled water. Various mixtures were tested to establish the best analytical conditions; in each mixture, the concentration of the particular component considered was varied, while the concentrations of other components were identical. The concentrations finally chosen for reagent solution C, were 0.2 M molybdate, 8.3 x lo-* M vanadate and 4.15 M sulfuric acid. Procedures Standard procedure for determining orthophosphate in water (Procedure A) and examining analytical conditions (Procedure C). Transfer up to 20 ml of sample solution to a 25-ml volumetric flask, and if necessary, dilute with distilled water to 20 ml. Add 3 ml of the reagent solution A (Procedure A) or reagent solution C (Procedure C) followed by 0.5 ml of the malachite green solution (5 x 10m3 M). Within 2 min of mixing the solutions, add 0.5 ml of PVA-217 solution (5%, w/v), then dilute to the mark with distilled water and mix. Measure the absorbance at 620 nm after standing for 10 min. Procedure for determining orthophosphate and totalphosphate in river waters (Procedure B). Transfer up to 20 ml of river water sample to a 25-ml volumetric flask, and if necessary, dilute with distilled water to 20 ml. Add 0.5 ml each of 9 M sulfuric acid solution and aqueous 40% (w/v) potassium peroxodisulfate solution, and heat in a water bath above 95 ‘C for 90 min. Cool to room temperature. Add 0.2 ml of sodium thiosulfate solution (10-l M) and stand for 10 min, if it is necessary to remove the interference from arsenic. Add 2 ml of reagent solution B and 0.5 ml of the malachite green solution (5 x 10m3 M). Within 2 min of mixing the solutions, add 0.5 ml of the PVA-217 solution and complete the determination as in Procedure A. For the determination of orthophosphate in river waters, transfer up to 20 ml of sample to a 25-ml volumetric flask and, if necessary, dilute with distilled
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water to 20 ml. Add 0.5 ml of 9 M sulfuric acid solution. Then continue as in Procedure B from the point of adding sodium thiosulfate. Procedure based on the formation of molybdophosphate (Procedure 0). Determine phosphate in a similar manner as the Procedure A, but with a reagent solution, not containing ammonium vanadate.
RESULTSANDDISCUSSION Composition of the reagent solution In a series of reagent solutions C, the molybdate concentration was varied from 1.2 x 10m2to 4.8~ 10m2M in the final solution, while the concentrations of vanadate and sulfuric acid in the reagent solution C were 8.3 x 10m4M and 4.15 M, respectively. The absorbance of the reagent blank increased gradually with increase in the amount of molybdate, probably because of phosphate contamination in the molybdate. Maximum absorbance was obtained in the range (2.4-3.0) x 10m2 M in the final solution. In this work, the final concentration of molybdate was adjusted to 2.4 x 10s2 M, i.e., the concentrations in reagent solutions A and B were fixed at 0.2 M and 0.3 M, respectively. In an analogous manner, the vanadate concentration was varied, while the concentrations of molybdate and sulfuric acid in reagent solution C were 0.2 M and 4.15 M, respectively. The results obtained are shown in Fig. 1. The absorbances of the reagent blank were almost constant (about 0.03). On addition of small amounts of vanadate, the absorbance decreased initially and gradually increased. These results were reproducible. In the range (0.42) x 10m4 M in the final solution, the absorbance was at a maximum. Thus the final concentration of vanadate chosen was about 1 x 10V4 M, i.e., the concentrations in reagent solutions A and B were fixed at 8.3 x 10m4 M and 1.25 X 10V3 M, respectively. In reagent solution C (0.2 M molybdate, 8.3 x 10e4 M) hydrochloric acid, nitric acid and perchloric acid were tested instead of sulfuric acid. The results obtained are shown in Table 1. The absorbances obtained with hydrochloric acid or sulfuric acid were in good agreement, whereas nitric acid and perchloric acid caused decreased absorbances. Sulfuric acid was preferred to hydrochloric acid, because it is difficult to prepare a reagent solution containing 8.3 M hydrochloric acid and because chloride reacts with peroxodisulfate, giving serious error in the determination of total phosphorus. The concentration of malachite green was varied from 2~ 10V5 M to 1.4~ 10m4 M. In the range (8-12)x 10m5 M in the final solution, the absorbance was maximal. Thus the final concentration of malachite green chosen was 1 x 10e4 M, i.e., the concentration in the stock solution was fixed at 5 X 10e3 M.
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0.5
g d $ B a 0.3
0.1
0.4
0.0 VW&ate
1.2
1.6
(10QM
2.0
0
lo
)
20
30
40 Time
50
60
(min)
Fig. 1. Effect of vanadate on the absorbance: ( 1,3 ) 4 x 10e6 M phosphate, (2 ) reagent blank. ( 1, 2) Measured against water; (3) measured against reagent blank. Concentrations refer to the final solutions. Fig. 2. Stability of the color in the absence and presence of stabilizing agents: (1) without stabilizingagent; (2), Brij-58 (0.1%); (3-8) PVA-217 (0.1%). (l-5) Unacidifiedmalachitegreen; (68) acidified malachite green solution. (1, 2, 3, 5, 6, and 8) 4~ lo-’ M phosphate in the final solution; (4,7) reagent blank. (l-4,6,7) Measured against water; (5,8) measured against reagent blank. TABLE 1 Comparison of sulfuric acid with other strong acids Acid
Absorbance’ Sample
Sulfuric acid, 0.5 M 0.532 Hydrochloric acid, 1.0 M 0.539
Reagent blank 0.046 0.050
Acid
Absorbance” Sample
Nitric acid, 1.0 M 0.355 Perchloric acid, 1.0 M 0.243
Reagent blank 0.032 0.029
“Obtained by Procedure A with unacidified malachite green solution for 4 x 10e6 M orthophosphatq water as reference.
Stabilizing agents The stability of the absorbance in the presence of surfactants was examined. The absorbance decreased with increasing time in the absence of PVA and in the presence of Brij-58 (Fig. 2). The other non-ionic surfactants (see Experimental) behaved similarly. In the presence of PVA-217, absorbances for solutions containing phosphorus and for the reagent blank decreased up to 20 min, but were then almost constant. The stabilizing effect of PVA-405 was almost the same as that of PVA-217. The amount of PVA added (in the range 0.02-
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0.2% w/v) had no significant effect on the reagent blank. For phosphate solutions, the absorbance increased gradually and subsequently decreased gradually with increasing amounts of PVA. The final concentration of PVA chosen was O.l%, i.e., the concentration of the stock solution was fixed at 5% (w/v). Figure 3 shows the effect of the time (t) of addition of the PVA solution. Absorbances at t= 0 were obtained by mixing the reagent, acidified malachite green and PVA solutions as quickly as possible; other absorbances were measured at different elapsed times from mixing the reagent and malachite green solutions for about 3 s to adding the PVA solution. All absorbances were measured 10 min after the addition of PVA. In the range 0.5-2 min, the absorbance of the reagent blank was at an almost constant maximum; therefore, in the recommended procedure, the PVA solution is added l-2 min after the sample, reagent and malachite green solutions have been mixed. Figure 2 shows the effect of standing time after the addition of the PVA on the absorbances. The acidified malachite green solution (curves 6-8) was preferable to the unacidified solution (curves 3-5 ); the absorbances became constant in a shorter time although they were slightly smaller. Absorption spectra and calibration graph
Figure 4 shows the absorption spectra which were obtained by Procedure A. The wavelength of maximum absorption was 620 nm; at this wavelength, the calibration graph was linear for O-l x 10m5 M phosphorus and the molar absorptivity was 1.05 X lo5 1mol-l cm-‘.
“‘“I
_ 0
,tas -
2
2
^
4
6 Time
(mtn)
8
10 Wavelength
/ “m
Fig. 3. Effect of time of addition of PVA solution: (1, 3) 4X lo-’ M phosphorus in the final solution; (2) reagent blank. (1,2) Measured against water; (3) measured against reagent blank. Fig. 4. Absorption spectra: (1) 4~ 10m6M phosphorus; (2) reagent blank. Reference: water.
125
Effect of diverse ions
The interference of diverse ions was examined by Procedure A; the results are shown in Table 2. The amounts of ions generally present in the river waters analyzed here are much smaller than those listed in Table 2. The interference of silicate ion is shown in Fig. 5 for the procedures based on the formation of molybdophosphate (Procedure D) and on the formation of vanadomolybdophosphate (Procedure A). Apparently, the interference is smaller in procedure A than in procedure D, probably because vanadomolybdosilicate is less likely to form. The interference in both procedures is much smaller than that reported previously [ 141, where even 5 x low5 M silicate interfered with the determination of phosphate. TABLE 2 Concentrations” of ions causing errors of < 1.5% the recovery of 4 x 10v6 M phosphorus by Procedure A Ion
Concentration/M
None Na+, ClK+, HCO, Ca*+, Mg*+ A13+ Fe3+, NH:, Ni*+, Co*+, Zn*+ NO,
10-l 5x10-2 10-Z 5x10-4 10-3 5x10-3
“Concentrations in the final solutions. Cations were added as their sulfate or chloride salts, and anions as their sodium or potassium salts. (10-M
Arsentc
I
0 Slllcate
(10-3M
1
2
3
4
8
12
)
Arsenic
1
4 1
5
16
20
I
24
(IO-'M)
Fig. 5. Effect of silicate: (0 ) procedure A; (0 ) procedure D. Fig. 6. Effect of arsenic: (1) Procedure A; (2,3) Procedure B, but without the addition of sodium thiosulfate; (4) Procedure B. Form of As added: (1,2,4) As(V); (3) As(II1). Concentrations are those in the final solutions.
126
Arsenic (V ) reacts in a similar manner to orthophosphate, as shown in Fig. 6. With Procedure A, 5 x 10m7M arsenic(V) increases the absorbance by 0.003. In Procedure B, by which total phosphorus is determined, arsenic (III) interferes to the same extent as arsenic(V) (Fig. 6)) because it is oxidized to arsenic (V) . Arsenic (V) , however, is easily reduced to arsenic (III) with sodium thiosulfate at pH c 1. The addition of 0.2 ml of 0.1 M sodium thiosulfate/ml of solution sufficed to reduce 5 x 10T5 M arsenic(V) to arsenic(II1). When more than 0.6 ml/ml was added, precipitation occurred. In Procedure B involving treatment with potassium peroxodisulfate and thiosulfate, the interference of 5x 10m6 M arsenic could be removed by adding 0.2 ml of 0.1 M thiosulfate (Fig. 6). Determination of phosphorus in river water
The precision of the method was evaluated by Procedure A. For replicate (7) determinations of 4 x 10m6 M phosphate, the relative standard deviation TABLE 3 Determination of phosphorus as orthophosphate and total phosphorus in river water samples Sample”
Phosphorus (fig 1-r) This workb P as PO:-
Zasu River
Zasu River
Zasu River
Nishi River
Asahi River Takahashi River Yoshii River
I II III I II III A B C I II III
21.8fO.l 36.4 f 0.4 81 fO.l 7.9fO.l 27.1 -I 0.2 71.8kO.2 26.6kO.2 38.6 f 1.2 33.1 f 0.7 12.3f0.3 3 +o 1 f0 5 f0.2 4 kO.2 8 +O.l
Extraction method” Total P
15.0+0.1 34.1 f 0.0 83.4 f 0.6 35.6f0.2 51.8 + 0.5 47.4 + 0.3 22.2 f 0.2 8.2 +_0.0 3 +1
P as PO;21.7fO.l 40.3fO.l 83.7 + 0.1 7.7fO.l 27.6 + 0.5 73.8f0.6 22.2 + 0.1 40.6 + 0.6 35.7 f 0.3 16.8f0.4 2.0f0.2 2.1* 0.1 6.0fO.l 4.720.1 6.8fO.l
Total P
17.9 + 0.2 39.8f0.2 88.4 f 0.0 36.4 + 0.2 54.1 f 0.2 53.3 f 0.4 27.7kO.l 6.9 + 0.2 7.4f0.4
“I-III denote the order of sampling downstream. Samples were taken at different times during January 1987. A-C denote samples taken at different times on the same day. bResults obtained by Procedure B, the mean values of three experiments. ‘Extraction-spectrophotometry with malachite green [ 2 1.
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was 1.0%; the standard deviation of the blank absorbance was < 0.001, which corresponds to 1 x lo-* M phosphate. By using, Procedure B without the addition of thiosulfate, phosphorus as orthophosphate and total phosphorus was determined in river waters. The results obtained are shown in Table 3. The phosphorus concentrations obtained by Procedure B showed good agreement with those obtained by extractionspectrophotometry [ 21.
REFERENCES
6 7 8 9 10 11 12 13 14 15 16 17 18
S. Motomizu, T. Wakimoto and K. Taei, Anal. Chim. Acta, 138 (1982) 329. S. Motomizu, T. Wakimoto and K. Teei, Taianta, 31 (1984) 235. K. Itaya and M. Ui, Clin. Chim. Acta, 14 (1966) 361. I. Steplnovi, Clin. Chim. Acta, 16 (1967) 330. A.J. Bastiaanse and C.A.M. Meigers, Z. Klin. Chem. Klin. Biochem., 6 (1968) 48 (Anal. Abstr., 17 (1969) 2872.) A.J. Bastiaanse and C.A.M. Meigers, Z. Klin. Chem. Klin. Biochem., 6 (1968) 109 (Anal. Abstr., 17 (1969) 2873.) W. Hohenwallner and E. Wimmer, Clin. Chim. Acta, 45 (1973) 169. A. Kallner, Clin. Chim. Acta, 59 (1975) 35. C.L. Penney, Anal. Biochem., 75 (1976) 201. B. Anner and M. Moosmayer, Anal. Biochem., 65 (1975) 305. D.J. Stewart, Anal. Biochem., 62 (1974) 349. H.J. Altmann, E. Ftirstenau, A. Gielewski and L. Scholz, Fresenius’ Z. Anal. Chem., 256 (1971) 274. A.G. Fogg, S. Soleymanloo and D.T. Burns, Anal. Chim. Acta, 88 (1977) 197. S. Motomizu, T. Wakimoto and K. T8ei, Analyst, 108 (1983) 361. S. Motomizu, T. Wakimoto and K. Taei, Talanta, 30 (1983) 333. K. Toei, M. Oshima and T. Kuwaki, Bunseki Kagaku, 34 (1985) 796. See, e.g., Z. Marczenko, Spectrophotometric Determination of Elements, 2nd edn., Horwood, Chichester, 1987. T. Fukumoto, K. Murata and S. Ikeda, Anal. Chem., 56 (1984) 929.
