ANALYTICA

CHIMICA ACTA
ELSEVIER
Analytica Chimica Acta 302

( 1095) 6Y-74

Determination of phosphorus in aqueous solution via formation of the phosphoantimonylmolybdenum blue complex Re-examination of optimum conditions for the analysis of phosphate
L. Drummond, W. Maher * IVater Research
Centre, University

of Canberra. PO Box 1. Balconnen, ACT 2616, Australia

received 2 August 1994

Received 5 April 1904; revised manuscript

Abstract
This paper describes an investigation of the conditions affecting the determination of phosphate using the reduced phosphoantimonylmolybdic acid method. The aim was to develop a determination method with faster kinetics than the original procedure of Murphy and Riley, for automation using flow-injection analysis. Optimum colour formation was found to occur at [H+ ]/ [MOO:-] ratios between SW30 at all pH values tested (0.36-1.06). The maximum rate of formation occurs at a [H ] / [MOO:- ] ratio of 70 within a pH range of 0.574.88 when an antimony concentration greater than 0.06 mM and ascorbic acid concentration greater than 0.009 M in the final solution are used. Full colour development occurs within 0.8-l min. The ascorbic acid reagent was found to be stable for 30 days. The results of the study indicated that by suitable selection of reagent conditions, rapid chromophore development can be achieved.
Kqwxfs: Phosporus; Phosphoantimonylmolybdic acid method

1. Introduction
The formation of 12-molybdophosphoric acid and reduction in the presence of antimony by either ascorbic acid or stannous chloride to the intensely coloured molybdenum blue followed by calorimetric quantification based on the original method of Murphy and Riley [ 11, is by far the most widespread method used for the determination of phosphorus in natural waters

12Jl.
Sb

PO-;- + 12MoO:PSbzMo,,O:;
* Corresponding

+PMo,,O:,

+ 1202-

Examination of the literature reveals that a wide range of concentrations of molybdate, pH, antimony and reductants are used (Table 1) As well the molar absorptivity of published methods vary widely (Table 1) as do details of reagent stability [ 1,2,4,5] and the time required for colour development [ 1,2,6-81. The primary aim of this study was to examine the optimum conditions under which phosphoantimonyl molybdenum blue is formed and develop a determination method with faster kinetics than the Murphy and Riley [ 11 procedure, with a view to the eventual application of this method in a flow injection mode.

author.

000.3.2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDIOOO3-2670(94)00429-3

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L. Drummond,

W. Maher / Analytica Chimica Acta 302 (1995) 69-74

Table 1 Molybdenum Ref. pH

Blue method; variations H+ (mM) 1080 400

in final reagent concentrations MOO:11.2 5.46 5.46 9.59 5.44 4.1 5.6 2.61 (mM)

and molar absorptivities Sb (mM)

a Ascorbic Acid (mM) 11.4 5 23 2.3 5.7 0.4 10.7 20,150 _ 22,700 Molar absorptivity 9574 17,935 21,700 -

H+ /MOO:71.4 73.3 73.3 70+10 73.5 73.5 73.3 76.6

[I61

rr1 [I41 f91 [I71 [41 [21 [71

a Assuming

- 0.03 0.4

_
0.07 0.32 0.82 0.04 0.06 0.054

0.4 0.22 0.4 0.5 0.4 0.7

400 600 400 300 400 200

50 ml sample.

2. Experimental
All chemicals were of analytical reagent grade. All glassware was cleaned by soaking in phosphate free detergent, acid washing and rinsing with distilled deionised water.

added. Absorbance of the reduced phosphoantimonylmolybdenum blue complex was measured at 880 nm (25C).

3. Results and discussion 3.1. Effect of [H+]I[Mo~~] The absorbance for 100 pg P/l solutions at various pH values is given in Fig. 1. The ascorbic acid and antimony concentrations in the final solution were 0.009 M and 0.06 mM respectively. The time of colour development was 2 min. The results presented are the mean of five replicates and the reproducibility as measured by the coefficient of variation was 0.2-3.3%. For

2.1. Instrumentation
A Hitachi Model U-3200 double beam spectrophotometer with 1 cm or 5 cm cuvettes was used for absorbance measurements.

2.2. Standards
A stock solution of phosphate (100 mg P/l) was prepared by dissolving 0.439 g of potassium dihydrogenphosphate in 1 1 of distilled deionised water. Phosphate working standards (50-100 pg P/l) were prepared daily by dilution. 2.3. Optimized reagents Mixed reagent: sulphuric acid (6.6 M), ammonium molybdate tetrahydrate (0.018 M) and potassium antimony tartrate (0.003 M). Reducing solution: ascorbic acid (0.5 M) in distilled deionised water. 2.4. Procedure To 25 ml of solution containing O-100 pg P/l, 0.5 ml of mixed reagent and 0.5 ml of ascorbic acid was

01

120

Tm--T---__

xi

T----r_ 70 80

[H+]/[MoO41

co

i.li05, 046 0 36

pH

,a

Fig. 1. Effect of

H+

] / [MOO: ] ratio on colour development.

L. Drummond, W. Maher IAnalytica Chimica Acta ,102 (1995) 69-74

71

Table 2 Absorbance Time of development

of blank at

[H ] I [MOO:- ] ratio of 20
PH

(min) 0.46 0.57 0.021* 0.003 0.026 + 0.003 0.76 0.062 + 0 007 0.260 * 0.03 0.8X 0.064 _t 0.007 0.139 + 0.02

I .Oh
0.056 + 0.00h 0.250 + 0.02 -__

?
10 Mean :t_S.D.. n = 4.

0.015 + 0.003 0.017 * 0.003

each pH there is a [ Ht ] / [MOO:- ] range which gives a constant analytical response. This range varies with pH but the ratio 45-80 gives a constant response at all the pH values tested (0.36-1.06). It is evident that as pH increases the range of constant response decreases. At [H ] / [MOO:- ] ratios < 20 the absorbance of the blank is high for all pH (Table 2). These results accord with previous studies 19,101 that [H+ ] / [MOO:- ] ratios of 50-80 give a plateau in which colour formation is complete but self reduction of molybdate ion does not occur. At higher ratios formation of molybdenum species which are unreactive with phosphate occurs. Stoichiometric studies have shown that MO(W) is predominantly dimerized in solutions of pH 0.3-0.9 [ 4,l l] and it is this form that reacts with orthophosphate [ 9,121. The ratio of Xl-80 corresponds to the region where the molybdenum dimer exists [ 91. It has been suggested that pH should be less than 0.7 to avoid the reduction of Mo(V1) to Mo( V). From Fig. 1 it is evident that at pH of 1.06 reduction is not occurring at [H ] / [MOO:- ] ratios :> 50 while at low pH values < 0.57 reduction is not occurring at [H+ ] / [ MOO:- ] above 40.

There appears to be no variation in A,;,,, i.e., 880 nm with [H]/[MoO~-1. A [Hf ] / [MOO:- ] ratio of 70 and pH 0.76 was used in all further experiments. 3.2. Effects of ascorbic acid The concentration of ascorbic acid was optimized to achieve the fastest reaction time and most economical use of the reagent. The antimony concentration in the final solution was 0.06 mM. Increasing the concentration of ascorbic acid to 0.009 M reduced the reaction time to 1.5 min (Fig. 2). Further addition of ascorbic acid did not increase the speed of reaction. Spectrophotometric studies of reduced molybdoantimonylphosphoric blue [9] indicate that an ascorbic acid concentration of at least twenty times the maximum phosphate level present is necessary to obtain full colour development within 10-30 min. It is evident from our results that a large excess is required to reach equilibrium rapidly. In all further experiments an ascorbic acid concentration of 0.009 M in the final solution was used. 3.3. Effect of antimony The antimony concentration was optimised for speed of reaction and precision of the blank. The shortest reaction time (Fig. 3) was achieved with an antimony concentration in the final solution greater than 0.03 mM. Further addition of antimony did not increase the speed of reaction. Acceptable precision of the blank (coefficient of variation < 10%) occurs when antimony concentrations greater than 0.06 mM are used. At lower antimony levels the precision of the blank is poor (coefficient of variation > 25%).

Time (min)

t\

lLx~r!ic tS
kid;M

I,

30

II

Fig. 2. Effect of ascorbic acid concentration on time of maximum colour development for a 100 /.~g P/l standard.

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L. Drummond, W. Maher IAnalytica Chimica Acta 302 (1995) 69-74

ent complex is formed with a lower absorption maximum [ 141. It is claimed in the literature that antimony acts as a catalyst [4,14]. Spectral studies have shown that antimony is present in the complex in a 2: 1 ratio and the stoichiometry of the reduced molybdoantimonylphosphoric acid is PSb,Mo,,O,, [ 91, while antimony may have a catalytic effect on reaction rate it does not act as a catalyst. 3.4. Stability of reduced
phosphoantimonylmolybdenum
Fig. 3. Effect of antimony concentration on the time of maximum colour development for a 100 pgll standard.

blue

The sensitivity of the analysis is decreased by the addition of antimony The regression lines for absorbance (Abs.) vs. phosphate concentration are Abs.= -O.OO1+3.5X1O-3 P, 12=0.9998 (A,,,= 880 nm; 30 min colour development) and Abs.= 0.003 + 4.32 X lop3 P, 4 = 0.9995 (h,,, = 820 nm; 12 h colour development), respectively, with and without antimony. The decrease in sensitivity is 17% at the 100 pg P/l level. The work of Murphy and Riley [l] who first used antimony, also shows a decrease in sensitivity, i.e., 10% for a 60 pg P/l solution. This is in contrast to Towns [ 41 who maintains that the addition of antimony results in higher sensitivity. deHaas et al. [ 131 have observed that increasing the concentration of antimony above that specified in Standard Methods [ 21 only increases the measured absorbance when phosphorus levels are greater than 1 mg/l probably because of increased amounts required to form the phosphoantimonylmolybdenum blue complex. In the presence of insufficient antimony a differ0.3

Time scans showed (Fig. 4a) that measurements must be made within 25 min to avoid loss of sensitivity. The addition of poly( vinyl alcohol) (0.04% in final solution) prevents loss in sensitivity (Fig. 4b). The mechanism of stabilization is unclear. 3.5. Reaction rate The rate of formation of the phosphoantimonylmolybdenum blue complex is dependent on the final [H+ ] / [MOO:-] ratio (Fig. 5). Ratios between 50 and 70 give the fastest reaction rates at all pH. For pH 0.360.88 full colour development occurs within 0.8-l min (Fig. 5). At pH 1.06 the time of colour formation increases to 1.5 min. The maximum rate of formation of the blank occurs at a [H+ ] / [MOO:-] ratio of 70 and a pH range of 0.57-0.76.
0.460.57
----1

130.76 DO.88 -__

fE41.061

0.2 r Absorbance

O.l-

o.oo

a 15

30 Time (mid

45

60 [H+]/[Mo04] ea ~CCJ

Fig. 4. Stability of reduced molybdoantimonylphosphoric acid (a) without poly(viny1 alcohol), (b) with poly( vinyl alcohol).

Fig. 5. Time required for development of maximum absorbance. pg P/l. Bar represents key to pH values.

100

L. Drummond,

W. Maher IAnalytica

Chimica Acta 302 (1995) 69-74

73

ooa-.

0.04

4
0 5 10 15 mu 20 vv~l 25 30 35

I 40

Fig. 6. Stability of ascorbic acid in glycerol, distilled deionized water and sulfuric acid.

Pai et al. [ lo] have shown that the time of maximum colour formation can be reduced to 40 s by raising the temperature to 70C. This is not desirable as it will also raise the rate of silicate reaction with the molybdate reagent [ 6,151. We have found that at room temperature (Xl-25C) silicate concentrations of up to 10,000 /Lg Sill can be present without causing any over-estimation of phosphorus ( < 5%). Silicate at the 100 mg Si/l level results in a 25% over-estimation of phosphate. Arsenate another common interferent does not cause an error when it is present at levels less than 10 pg As/l. 3.6. Stability of reagents Acidified molybdate containing antimony but without ascorbic acid was stored in a brown glass bottle at 4-6C and allowed to return to room temperature before use. It was found to be stable for at least six months. Other studies have reported that the acidified molybdate reagent is only stable for 4-24 h [ 1,2,6]. The stability of ascorbic acid in water, sulfuric acid and glycerol at various concentrations (10, 25, 50, 75 and 100%) were also investigated (Fig. 6). The reduc-

tants were stored in clear glass bottles exposed to light and kept at ambient temperature (20-25C) for 40 days. Ascorbic acid in 100% glycerol was too viscous to use and was not investigated further. Ascorbic acid in glycerol ( lO-75%), water and sulphuric acid (0.36 M) were stable for 27, 18 and 15 days respectively (Fig. 6). Ascorbic acid has been reported in the literature to be stable in distilled water and sulfuric acid (0.1-0.5 M) for up to 7 days [ 2,7] and in 10% glycerol for 30 days [ 81. The reasons for the longer periods of stability in distilled water and sulfuric acid found in this study are unclear but may be related to initial concentration and purity of the ascorbic acid solution.

4. Conclusion This study was performed to determine the optimal conditions for measuring phosphate at levels up to 100 /Lg P/l, levels typically measured in oligotrophic and eutrophic Australian natural waters. By selecting a [H+ ] / [MOO:-] ratio of 70 and a pH 0.57-0.88 in the final solution together with an excess of antimony (0.06 mM) and ascorbic acid (0.009 M), full colour devel-

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L. Drummond,

W. Maher IAnalytica

Chimica Acta 302 (1995) 69-74

opment can be achieved within 0.8-l min. Reagents have also been found to be stable for 27 days if ascorbic acid is prepared in lO-70% glycerol solution. It has been shown at higher phosphate concentrations than about 800 pg/l, and using similar reagent concentrations to those here, that incomplete colour development occurs and a change in absorption maximum results unless additional antimony is added [ 4,141. The optimized conditions described here therefore may not be applicable to highly eutrophied systems or waste waters containing phosphorus levels in excess of 800 pg P/l.

References
[l] J. Murphy and J. P. Riley, Anal. Chim. Acta, 27 (1962) 31. [Z] L.S. Clesceri, A.E. Greenberg and R.R. Trussell (Eds.), Standard Methods for the Examination of Water and Waste Water, 17th edn., APHA-AWWA-WPC, 1989, pp. 4-166. [ 31 0. Broberg and K. Pettersson, Hydrobiologia, 170 (1988) 45. [4] T.G. Towns, Anal. Chem., 58 ( 1986) 223.

[5] C. Ciavatta, L.V. Antisari and P. Sequi, J. Environ. Qual., 19 (1990) 761. [6] L.J. Lennox, Water Res., 13 (1979) 1329. [ 71 K. Grasshoff, in Methods of Seawater Analysis, Determination of Nutrients, Verlag Chemie, Weinhem, 1976, Chap. 9, pp. 117-126. [8] P.J. Worsfold, J.R. Clinch and H. Casey, Anal. Chim. Acta, 197 (1987) 43. [ 91 J.E. Going and S.J. Eisenreich, Anal. Chim. Acta, 70 (1974) 95. [lo] S.C. Pai, CC. Yang and J.P. Riley, Anal. Chim. Acta, 229 (1990) 115. [ 111 S.R. Crouch and H.V. Malmstadt, Anal. Chem., 39 (1967) 1084. [12] A.C. Javier, S.R. Crouch and H.V. Malmstadt, Anal. Chem., 40 (1968) 1922. [ 131 D.W. deHaas, L.H. Lotter and LA. Dubery, Water SA, 16 (1990) 55. [ 141 J.E. Harwood, R.A. VanSteenderen and A.L. Kuhn, Water Res., 3 (1969) 417. [15] K.I. Aspila, H. Agemian and A.S.Y. Chau, Analyst, 101 (1976) 187.

[ 161 D.N. Fogg and N.T. Wilkinson, Analyst, 83 (1958) 406. [ 171 J.T.H. Goossen and J.G. KJoosterboer,Anal. Chem.,50 (1978)
707.