Analytica Chimica Acta, 211 (1988) 119-127 119

Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

SPECTROPHOTOMETRIC DETERMINATION OF PHOSPHORUS

IN RIVER WATER BASED ON THE REACTION OF VANADO-
MOLYBDOPHOSPHATE 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 spe-
cies either as they are or after extraction into an organic solvent as a proton-
ated species as an ion associate with a bulky cation. Motomizu et al. [ 1,2] have
reported sensitive extraction-spectrophotometric methods for the determina-
tion 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 ne-
cessity for extraction. Itaya and Ui [ 31 reported a simple and sensitive spec-
trophotometric 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

0003-2670/88/$03.50 0 1988 Elsevier Science Publishers B.V.

120

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 mala-
chite 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 molybdophos-
phate but the sensitivity provided is less than that obtained with phospho-
molybdenum. 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 phospho-
rus in river water were studied.

EXPERIMENTAL

Apparatus and reagents

Absorption measurements were made with a Hitachi Model 139 spectropho-
tometer 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 commer-
cially 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 com-
pound (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
 121

217 and 405 (number average degree of polymerization 1700 and 500, respec-
tively) 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 van-
adate solution, 22.8 ml of concentrated sulfuric acid and 28.9 ml of distilled
water. Reagent solution B (for determining orthophosphate and total phos-
phate in river waters) was prepared by mixing 60 ml of 0.5 M molybdate so-
lution, 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 con-
centrations 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 dis-
tilled 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 tem-
perature. 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
122

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). De-
termine 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 con-
tamination 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 ad-
dition of small amounts of vanadate, the absorbance decreased initially and
gradually increased. These results were reproducible. In the range (0.4-
2) 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 concen-
trations 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 hy-
drochloric 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 absor-
bance 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.
 123

0.5

g
d
$
B
a 0.3

0.1

0.4 0.0 1.2 1.6 2.0 0 lo 20 30 40 50 60

VW&ate (10QM ) Time (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 stabi-
lizingagent; (2), Brij-58 (0.1%); (3-8) PVA-217 (0.1%). (l-5) Unacidifiedmalachitegreen; (6-
8) 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 Acid Absorbance

Sample Reagent blank Sample Reagent blank

Sulfuric acid, 0.5 M 0.532 0.046 Nitric acid, 1.0 M 0.355 0.032
Hydrochloric acid, 1.0 M 0.539 0.050 Perchloric acid, 1.0 M 0.243 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 Experi-
mental) behaved similarly. In the presence of PVA-217, absorbances for solu-
tions 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-
124

0.2% w/v) had no significant effect on the reagent blank. For phosphate so-
lutions, the absorbance increased gradually and subsequently decreased grad-
ually 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 mea-
sured at different elapsed times from mixing the reagent and malachite green
solutions for about 3 s to adding the PVA solution. All absorbances were mea-
sured 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 pref-
erable to the unacidified solution (curves 3-5 ); the absorbances became con-
stant 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 ab-
sorptivity was 1.05 X lo5 1mol-l cm-.

_ 2 ^
,tas - -
0 -
2 4 6 8 10
Time (mtn) 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 vanadomolyb-
dosilicate 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 Pro-
cedure A

Ion Concentration/M
None
Na+, Cl- 10-l
K+, HCO, 5x10-2
Ca*+, Mg*+ 10-Z
A13+ 5x10-4
Fe3+, NH:, Ni*+, Co*+, Zn*+ 10-3
NO, 5x10-3

Concentrations in the final solutions. Cations were added as their sulfate or chloride salts, and
anions as their sodium or potassium salts.

Arsentc (10-M 1
1 2 3 4 5
I 1 I

0 4 8 12 16 20 24
Slllcate (10-3M ) Arsenic (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) inter-
feres to the same extent as arsenic(V) (Fig. 6)) because it is oxidized to ar-
senic (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 in-
volving treatment with potassium peroxodisulfate and thiosulfate, the inter-
ference 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 Extraction method

P as Total P P as Total P
PO:- PO;-

Zasu River I 21.8fO.l 21.7fO.l

II 36.4 f 0.4 40.3fO.l
III 81 fO.l 83.7 + 0.1
Zasu River I 7.9fO.l 15.0+0.1 7.7fO.l 17.9 + 0.2
II 27.1 -I 0.2 34.1 f 0.0 27.6 + 0.5 39.8f0.2
III 71.8kO.2 83.4 f 0.6 73.8f0.6 88.4 f 0.0
Zasu River A 26.6kO.2 35.6f0.2 22.2 + 0.1 36.4 + 0.2
B 38.6 f 1.2 51.8 + 0.5 40.6 + 0.6 54.1 f 0.2
C 33.1 f 0.7 47.4 + 0.3 35.7 f 0.3 53.3 f 0.4
Nishi River I 12.3f0.3 22.2 f 0.2 16.8f0.4 27.7kO.l
II 3 +o 8.2 +_0.0 2.0f0.2 6.9 + 0.2
III 1 f0 3 +1 2.1* 0.1 7.4f0.4
Asahi River 5 f0.2 6.0fO.l
Takahashi River 4 kO.2 4.720.1
Yoshii River 8 +O.l 6.8fO.l

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 mala-
chite green [ 2 1.
 127

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 re-
sults obtained are shown in Table 3. The phosphorus concentrations obtained
by Procedure B showed good agreement with those obtained by extraction-
spectrophotometry [ 21.

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