1995 drummond maher determination of phosphorus

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ELSEVIER

ANALYTICACHIMICAACTA

Analytica Chimica Acta 302 ( 1095) 6Y-74

Determination of phosphorus in aqueous solution via formation ofthe phosphoantimonylmolybdenum blue complex

Re-examination of optimum conditions for the analysis ofphosphate

L. Drummond, W. Maher *IVateresearch Centre, University ofCanberra. PO Box 1. Balconnen, ACT 2616, AustraliaReceived 5 April 1904; revised manuscript received 2 August 1994

AbstractThis paper describes an investigation of the conditions affecting the determination of phosphate using the reduced phosphoan-

timonylmolybdic acid method. The aim was to develop a determination method with faster kinetics than the original procedureof 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 acidconcentration greater than 0.009 M in the final solution are used. Full colour development occurs within 0.8-l min. The ascorbicacid 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. IntroductionThe formation of 12-molybdophosphoric acid and

reduction in the presence of antimony by either ascorbicacid or stannous chloride to the intensely colouredmolybdenum blue followed by calorimetric quantifi-cation based on the original method of Murphy andRiley [ 11, is by far the most widespread method usedfor the determination of phosphorus in natural waters12Jl.

SbPO-;- + 12MoO:- +PMo,,O:, + 1202- +

PSbzMo,,O:;

Examination of the literature reveals that a widerange of concentrations of molybdate, pH, antimonyand reductants are used (Table 1) As well the molarabsorptivity of published methods vary widely (Table1) as do details of reagent stability [ 1,2,4,5] and thetime required for colour development [ 1,2,6-81.

The primary aim of this study was to examine theoptimum conditions under which phosphoantimonylmolybdenum blue is formed and develop a determi-nation method with faster kinetics than the Murphy andRiley [ 11 procedure, with a view to the eventual appli-cation of this method in a flow injection mode.

* Corresponding author.

000.3.2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reservedSSDIOOO3-2670(94)00429-3


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70

Table 1

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

Molybdenum Blue method; variations in final reagent concentrations and molar absorptivities aRef. pH H+ (mM) MOO:- (mM) H+ /MOO:- Sb (mM) Ascorbic Acid (mM) Molar absorptivity

[I61 - 0.03 1080 11.2 71.4 _ 11.4 9574rr1 0.4 400 5.46 73.3 0.07 5 17,935[I41 0.4 400 5.46 73.3 0.32 23 21,700f91 0.22 600 9.59 70+10 0.82 2.3 -[I71 0.4 400 5.44 73.5 0.04 5.7[41 0.5 300 4.1 73.5 20,150[21 0.4 400 5.6 73.3 0.06 0.4 _[71 0.7 200 2.61 76.6 0.054 10.7 22,700a Assuming 50 ml sample.

2. ExperimentalAll chemicals were of analytical reagent grade. All

glassware was cleaned by soaking in phosphate freedetergent, acid washing and rinsing with distilleddeionised water.2.1. Instrumentation

A Hitachi Model U-3200 double beam spectropho-tometer with 1 cm or 5 cm cuvettes was used for absorb-ance measurements.2.2. Standards

A stock solution of phosphate (100 mg P/l) wasprepared by dissolving 0.439 g of potassium dihydro-genphosphate in 1 1 of distilled deionised water. Phos-phate working standards (50-100 pg P/l) wereprepared daily by dilution.2.3. Optimized reagents

Mixed reagent: sulphuric acid (6.6 M), ammoniummolybdate tetrahydrate (0.018 M) and potassium anti-mony tartrate (0.003 M). Reducing solution: ascorbicacid (0.5 M) in distilled deionised water.2.4. Procedure

To 25 ml of solution containing O-100 pg P/l, 0.5ml of mixed reagent and 0.5 ml of ascorbic acid was

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

3. Results and discussion3.1. Effect of [H+]I[Mo~~]

The absorbance for 100 pg P/l solutions at variouspH values is given in Fig. 1. The ascorbic acid andantimony concentrations in the final solution were0.009 M and 0.06 mM respectively. The time of colourdevelopment was 2 min. The results presented are themean of five replicates and the reproducibility as meas-ured by the coefficient of variation was 0.2-3.3%. For

011- 05, pH0460 20 Tm--T---__ i.li-36xi w 70 T----r_80[H+]/[MoO41 co ,a

Fig. 1. Effect of [ H+ ] / [MOO: ] ratio on colour development.


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Table 2

L. Drummond, W. M aher IA nalyt ica Chimi ca Acta ,102 1995) 69-74 71

Absorbance of blank at [H ] I [MOO:- ] ratio of 20 Time ofdevelopment (min) PH

0.46 0.57 0.76 0.8X I .Oh?

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

0.015 + 0.003 0.021* 0.0030.017 * 0.003 0.026 + 0.003

each pH there is a [ Ht ] / [MOO:- ] range which givesa constant analytical response. This range varies withpH but the ratio 45-80 gives a constant response at allthe pH values tested (0.36-1.06). It is evident that aspH increases the range of constant response decreases.At [H ] / [MOO:- ] ratios < 20 the absorbance of theblank is high for all pH (Table 2).

These results accord with previous studies 19,101that [H+ ] / [MOO:- ] ratios of 50-80 give a plateau inwhich colour formation is complete but self reductionof molybdate ion does not occur. At higher ratios for-mation of molybdenum species which are unreactivewith phosphate occurs. Stoichiometric studies haveshown that MO(W) is predominantly dimerized insolutions of pH 0.3-0.9 [ 4,l l] and it is this form thatreacts with orthophosphate [ 9,121. The ratio of Xl-80corresponds to the region where the molybdenumdimer exists [ 91. It has been suggested that pH shouldbe less than 0.7 to avoid the reduction of Mo(V1) toMo( V). From Fig. 1 it is evident that at pH of 1.06reduction is not occurring at [H ] / [MOO:- ] ratios:> 50 while at low pH values < 0.57 reduction is notoccurring at [H+ ] / [MOO:- ] above 40.

BTime (min) t\

0 2 4 lLx~r!icdM x tS I, 30 IIFig. 2. Effect of ascorbic acid concentration on time of maximumcolour development for a 100 /.~g P/l standard.

0.062 + 0 007 0.064 _t 0.007 0.056 + 0.00h0.260 * 0.03 0.139 + 0.02 0.250 + 0.02-__

There appears to be no variation in A,;,,, i.e., 880 nmwith [H]/[MoO~-1.

A [Hf ] / [MOO:- ] ratio of 70 and pH 0.76 wasused in all further experiments.

3.2. Effects of ascorbic acid

The concentration of ascorbic acid was optimized toachieve the fastest reaction time and most economicaluse of the reagent. The antimony concentration in thefinal solution was 0.06 mM. Increasing the concentra-tion of ascorbic acid to 0.009 M reduced the reactiontime to 1.5 min (Fig. 2). Further addition of ascorbicacid did not increase the speed of reaction. Spectropho-tometric studies of reduced molybdoantimonylphos-phoric blue [9] indicate that an ascorbic acidconcentration of at least twenty times the maximumphosphate level present is necessary to obtain full col-our development within 10-30 min. It is evident fromour results that a large excess is required to reach equi-librium rapidly.

In all further experiments an ascorbic acid concen-tration of 0.009 M in the final solution was used.

3.3. Effect of antimony

The antimony concentration was optimised for speedof reaction and precision of the blank.

The shortest reaction time (Fig. 3) was achievedwith an antimony concentration in the final solutiongreater than 0.03 mM. Further addition of antimonydid not increase the speed of reaction. Acceptable pre-cision of the blank (coefficient of variation < 10%)occurs when antimony concentrations greater than 0.06mM are used. At lower antimony levels the precisionof the blank is poor (coefficient of variation > 25%).


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72 L. Drummond, W. Maher I Analytica Chimica Acta 302 (1995) 69-74

Fig. 3. Effect of antimony concentration on the time of maximumcolour development for a 100 pgll standard.

The sensitivity of the analysis is decreased by theaddition of antimony The regression lines for absorb-ance (Abs.) vs. phosphate concentration areAbs.= -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; 12h colour development), respectively, with and withoutantimony. The decrease in sensitivity is 17% at the 100pg P/l level. The work of Murphy and Riley [l] whofirst used antimony, also shows a decrease in sensitiv-ity, i.e., 10% for a 60 pg P/l solution.

This is in contrast to Towns [ 41 who maintains thatthe addition of antimony results in higher sensitivity.deHaas et al. [ 131 have observed that increasing theconcentration of antimony above that specified in Stan-dard Methods [ 21 only increases the measured absorb-ance when phosphorus levels are greater than 1 mg/lprobably because of increased amounts required toform the phosphoantimonylmolybdenum blue com-plex. In the presence of insufficient antimony a differ-

0.3

0.2rAbsorbance

O.l-b

. a

o.oo a I t t15 30 45 60Time (mid

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

ent complex is formed with a lower absorptionmaximum [ 141. It is claimed in the literature that anti-mony acts as a catalyst [4,14]. Spectral studies haveshown that antimony is present in the complex in a 2: 1ratio and the stoichiometry of the reduced molybdoan-timonylphosphoric acid is PSb,Mo,,O,, [ 91, whileantimony may have a catalytic effect on reaction rate itdoes not act as a catalyst.3.4. Stabi li ty of reducedphosphoantimonylmolybdenum blue

Time scans showed (Fig. 4a) that measurementsmust be made within 25 min to avoid loss of sensitivity.The addition of poly( vinyl alcohol) (0.04% in finalsolution) prevents loss in sensitivity (Fig. 4b). Themechanism of stabilization is unclear.3.5. Reaction rate

The rate of formation of the phosphoantimonylmo-lybdenum blue complex is dependent on the final[H+ ] / [MOO:-] ratio (Fig. 5). Ratios between 50 and70 give the fastest reaction rates at all pH. For pH 0.36-0.88 full colour development occurs within 0.8-l min(Fig. 5). At pH 1.06 the time of colour formationincreases to 1.5 min.The maximum rate of formation of the blank occursat a [H+ ] / [MOO:-] ratio of 70 and a pH range of0.57-0.76.

----10.460.57 130.76 DO.88 fE41.061-__

[H+]/[Mo04] ea ~CCJ

Fig. 5. Time required for development of maximum absorbance. 100pg P/l. Bar represents key to pH val ues.


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L. Drummond, W. Maher IAnalytica Chimica Acta 302 (1995) 69-74 73

ooa-.

0.04 4 I0 5 10 15 20 25 30 35 40

mu vv~lFig. 6. Stability of ascorbic acid in glycerol, distilled deionized water and sulfuric acid.

Pai et al. [ lo] have shown that the time of maximumcolour formation can be reduced to 40 s by raising thetemperature to 70C. This is not desirable as it will alsoraise the rate of silicate reaction with the molybdatereagent [ 6,151. We have found that at room tempera-ture (Xl-25C) silicate concentrations of up to 10,000/Lg Sill can be present without causing any over-esti-mation of phosphorus ( < 5%). Silicate at the 100 mgSi/l level results in a 25% over-estimation of phos-phate. Arsenate another common interferent does notcause an error when it is present at levels less than 10pg As/l.

3.6. Stability of reagentsAcidified molybdate containing antimony but with-

out ascorbic acid was stored in a brown glass bottle at4-6C and allowed to return to room temperature beforeuse. It was found to be stable for at least six months.Other studies have reported that the acidified molyb-date reagent is only stable for 4-24 h [ 1,2,6].

The stability of ascorbic acid in water, sulfuric acidand glycerol at various concentrations (10, 25, 50, 75and 100%) were also investigated (Fig. 6). The reduc-

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

4. ConclusionThis study was performed to determine the optimal

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


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

opment can be achieved within 0.8-l min. Reagentshave also been found to be stable for 27 days if ascorbicacid is prepared in lO-70% glycerol solution.

It has been shown at higher phosphate concentrationsthan about 800 pg/l, and using similar reagent concen-trations to those here, that incomplete colour develop-ment occurs and a change in absorption maximumresults unless additional antimony is added [ 4,141.

The optimized conditions described here thereforemay not be applicable to highly eutrophied systems orwaste waters containing phosphorus levels in excess of800 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 WasteWater, 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, Determinationof 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, WaterRes., 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.

1995 drummond maher determination of phosphorus

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