Spectrophotometric Determination of Low Levels of the Orthophosphate Anion as Molybdenum Blue Using Sodium Thiosulphate Reducing Agent

Introduction

Phosphorus (P) a vital nutrient to human, animal and plant growth occurs naturally in rocks and soils as phosphorite or hydroxyapatite, Ca₃(PO4)₂.Ca(OH)₂, apatite, Ca₃(PO₄)₂.CaF₂, vivianite, Fe₃(PO₄)₂.8H₂O and aluminium phosphate¹. Since P serves as a nutrient in the food chain, supplementing the native P in the soil and animals’ diet where it is lacking may be a necessity. This can result in excess P moving from agricultural areas especially where animal wastes are being used as fertilisers to other environmental systems². These phosphates stimulate the growth of phytoplankton and aquatic plants which provide food for many organisms including zooplanktons, fish, humans and other mammals. This may result in eutrophication of waters and its related effects of anoxic conditions^3,4,5; which leads to deterioration in water quality and depletion of aquatic species. Phosphorus in freshwaters ranges from 0.005 to 0.02 mg L^-1PO₄-P in most natural surface waters, 0.001 mg L^-1 PO₄-P in some pristine waters and as high as 200 mg L^-1PO₄-P in some enclosed saline waters. Average groundwater levels are about 0.02 mg L^-1PO₄-P^6,7. Due to a few absorbing surfaces and constant mixing in water bodies, low P levels are responsible for increasing biological productivity making the aquatic system highly sensitive to P contamination. Some of the common sources of P into the environment include phosphate-containing geologic formations, agricultural fertilizer application, detergent usage and industrial wastes^8,9,10. This calls for constant monitoring of P in water systems and other vulnerable environmental systems.

Several forms of P including orthophosphate (reactive phosphate), condensed phosphate (pyro-, meta- and polyphosphate) and organic phosphate may exist in natural waters¹¹. These P forms can be determined either partially or fully as the orthophosphate which is the most thermodynamically stable phosphate form¹². Various analytical techniques have been reported for the determination of phosphate concentrations in water systems including titrimetric, complexogravimetric, atomic absorption spectrophotometry¹³, high performance liquid chromatography¹⁴, colorimetry^15,16, and UV-Vis spectrophotometric methods^17,18.Among the popular spectrophotometric methods of orthophosphate determination is the molybdenum blue¹⁹, and the yellow vanadomolybdate complex methods; of which the most commonly used is the molybdenum blue method due to its high sensitivity²⁰.This method involves condensation of molybdate with orthophosphate in aqueous acidic medium forming 12-molybdophosphoric acid which is selectively reduced to the phosphomolybdate anion (molybdenum blue) as shown in equations 1 and 2. The intensity of the blue colouredheteropoly compound is proportional to the amount of P present in the sample.

The molybdenum blue method since its introduction by Dickman and Bray, 1940²¹ has undergone several modifications to improve sensitivity, precision and selectivity for orthophosphate determination. Notably, improvements have been attempted through the use of various reductants such astin(II) chloride²², ascorbic acid²³, hydrazine sulphate, sodium sulphide¹⁸ and hydroquinone²⁴. However, most of the modified methods have disadvantages such as instability of the heteropoly blue colour, interference from arsenic and copper, the lengthy time required for full colour development, low sensitivity coupled with high absorption by the blank²⁵. The rate of colour development is specifically slow with ascorbic acid, the most commonly used reductant employed in the Murphy and Riley, 1962 method¹², whereas hydrazine sulphate another preferred reductant is toxic and corrosive²⁶. Besides, some of the modified methods are cumbersome and involve lengthy extraction procedures which require additional analyst skills^18,27. Therefore, the use of sodium thiosulphate was explored as a reductant.

In this study, we demonstrate the determination of low levels of the orthophosphate anion as molybdenum blue using sodium thiosulphatethe reducing agent. The sodium thiosulphate used to reduce molybdophosphoric acid to molybdenum blueis commercially available, cheap and a common laboratory reagentused in several redox reactions. It has been used in molybdenum blue reactions to counter arsenate interference by reducing As(V) to As(III)²⁸but it has not beenoptimized and reported for the orthophosphate determination. Moreover, the sodium thiosulphate solution is stable for several days as opposed to ascorbic acid the most commonly used reductant.

Materials and Methods

Materials

All chemicals were of analytical grade and used without further purification. They included; sodium thiosulphatepentahydrate (Merck, RSA, 99.0 – 101.0%), potassium dihydrogen phosphate (Merck, RSA  99.5%), hydrochloric acid (Merck, RSA,  32%), ammonium molybdatetetrahydrate (Sigma-Aldrich, RSA, ≥ 99%), antimony potassium tartrate (Hopkin and Williams, England), L-ascorbic acid (BDH, England 99.0 – 100.5 %), glacial acetic acid (Merck, RSA, 98 %), sulphuric acid, sodium carbonate anhydrous, starch soluble (BDH, England), potassium iodate (Merck, RSA, 99.0 – 101.0%), oxalic acid, potassium chloride, sodium sulphate (May and Baker Ltd, England), copper metal (Trust chemical laboratories, 99.5%), sodium arsenate, lead(II) nitrate and sodium nitrite.

Instrumental Analysis

The weighing was carried out using RADWAG WagiElektroniczne analytical balance (Model AS220.R2). The UV/Vis spectra were scanned using UV-VIS-NIR spectrophotometer (Shimadzu UV-3600) with 1 cm quartz cuvettes. The scanning wavelength was in the range of 500 – 1100 nm.

Preparation of Solutions

Deionized water was used to prepare all solutions.

Stock and working standard orthophosphate solutions

Stock orthophosphate solution was prepared by dissolving KH₂PO₄ (0.4393 g) in deionized water to make a solution of 0.1 mg P mL^-1. The working orthophosphate standard solution (0.01 mg P mL^-1) was prepared by appropriately diluting the stock phosphate solution.

Sulphuric acid solution

Sulphuric acid solution (0.5 M, 2.5 M and 5.5 M) was prepared by appropriately diluting concentrated H₂SO₄ (18.38 M).

Ethanoic Acid Solution

Ethanoic acid (0.5 M) was prepared by appropriately diluting glacial acetic acid (17.398 M).

Sodium Thiosulphate Solution

Sodium thiosulphate (Na₂S₂O₃.5H₂O, 7.50 g) and Na₂CO₃ (0.0301 g) were dissolved in 250 mL of recently boiled and cooled deionized water. The solution was standardized with KIO₃ as follows: KI (2.00 g) was dissolved in deionized water (25 mL) and HCl (6 M, 2 mL) added followed by KIO₃ (0.120 g). The liberated iodine solution was then titrated with Na₂S₂O₃ solution until the triiodide complex was less intense. Starch indicator (5 mL) was added and the titration continued until a dark blue colour of the starch-triiodide complex turned colourless. The titration was repeated three times.

Ascorbic Acid Solution

Ascorbic acid (C₆H₈O₆, 1.760 g) was dissolved in deionized water (100 mL) to give a concentration of 0.1 M.

Ammonium molybdate solution

Ammonium molybdate,(NH₄)₆Mo₇O₂₄ .4H₂O (4.00 g) was dissolved in deionized water (100 mL) to make a 0.0324 M solution.

Antimony potassium tartrate solution

Antimony potassium tartrate(KSbO.C₄H₄O₆, 0.2740 g) was dissolved in deionized water (100 mL) to make an 8.4326 x 10^-3 M solution.

Ammonium molybdate- antimonyl tartrate (AM-PAT)solution

The ammonium molybdate-antimonylpotassium tartrate (AM-PAT) solution was prepared by dissolving (NH₄)₆Mo₇O₂₄ .4H₂O (2.00 g) and KSbO.C₄H₄O₆ (0.050 g) in deionized water (250 mL). The AM-PAT solution was 6.473 x 10^-3 M in ammonium molybdate and 6.1552 x 10^-4 M in antimonyl potassium tartrate.

Combined reagent

Antimonyl potassium tartrate solution (8.4326 x 10^-3 M, 5 mL) was added to dilute H₂SO₄ (2.5 M, 50 mL) in a beaker followed by ammonium molybdate solution (0.0324 M, 15 mL) and ascorbic acid solution (0.1 M, 30 mL).

Orthophosphate anion analytical method development

To 50 mL volumetric flask was added the working orthophosphate solution, KH₂PO₄ (0.05 mg P mL^-1, 1mL), AM-PAT solution (4 mL), sulphuric acid (5.5 M, 1 mL) and Na₂S₂O₃ solution (0.1192 M, 1 mL). The solutions were made to the mark with deionized water.  The solution was allowed to stand for 10 minutes and then the spectrum scanned against deionized water as a blank in the range 500 – 1100 nm. Based on the spectrum, a maximum absorption (λ_max) of the reduced phosphopolyoxomolybdate complex was utilized in the subsequent analysis.

Optimisation of the sodium thiosulphate molybdenum blue method

Experimental parameters such as the concentration of various reagents and their orderof addition were studied separately and the absorbance of the coloured product observed at 880 nm. The optimization of the different parameters is presented in the proceeding sections.

Determination of concentration of ammonium molybdate – potassium antimony tartrate for use

In this setup, a varying amount of the AM-PAT solution was utilized to study the effect of concentration of ammonium molybdate on theformation of phosphomolybdenum blue (PMB) complex in the presence of potassium antimony tartrateas a catalyst.To three separate 50 mL volumetric flasks was added KH₂PO₄ solution (0.05 mg P mL^-1,1 mL), AM-PAT solution (2 – 10 mL), followed by H₂SO₄ (5.5 M, 1 mL) and Na₂S₂O₃ solution (0.1192 M, 1 mL). The solutions were made to the mark with deionized water, allowed to stand for10 minutes and the absorbance of the resultant molybdenum blue complex measured at 880 nm against a reagent blank.

Determination of concentration of the acid for use

To each of three separate 50 mL volumetric flasks was added KH₂PO₄ solution (0.05 mg P mL^-1,1 mL), an optimized volume of AM-PAT solution, sulphuric acid (5.5M, 0.25 – 2 mL) and Na₂S₂O₃ solution (0.1192 M, 1 mL). The solutions were diluted to the mark with deionized water, allowed to stand for 10 minutes and the absorbance of the molybdenum blue complex measured at 880 nm against a reagent blank.

Following the same procedure used for H₂SO₄, HCl and CH₃COOH were also used to determine the acid to be used.The different solutions were allowed to stand for 10 minutes and the absorbance of the resultant molybdenum blue complexes measured at 880 nm.

Influence of concentration of sodium thiosulphatereducing agent on molybdenum blue formation

The influence of the concentration of Na₂S₂O₃ reducing agent on the absorbance of the molybdenum blue complex was studied byutilizing varying volumes of the reductant.  To three separate volumetric flasks was added KH₂PO₄ solution (0.05 mg P mL^-1,1 mL) followed by the optimized volumes of AM-PAT and H₂SO₄ and the reducing agent Na₂S₂O₃ (0.1192 M, 0.25 – 2 mL). The solutions were made to the mark with deionized water, allowed to stand for 10 minutes and the absorbance measured at 880 nm against a reagent blank.

Order of Reagent Addition

The effect of the order of addition of the reagents on the absorbance of the molybdenum blue complex was studied using optimized volumes of different reagents. The addition order of different solutions including KH₂PO_4, AM-PAT, H₂SO₄ and Na₂S₂O₃ was varied and the absorbance of the resultant molybdenum blue complex measured at 880 nm.

Colour stability of reduced phosphopolyoxomolybdate complexes

The colour stability of the reduced phosphomolybdate complex was studied using optimized volumes and addition order of the reagents. To three different 50 mL volumetric flasks was added KH₂PO₄ solution (0.05 mg P mL^-1,1 mL) followed by addition of the colour developing agent comprised of AM-PAT, H₂SO₄ and Na₂S₂O_3. The absorbance of the resultant molybdenum blue complex was measured at 880 nm against a reagent blank immediately after mixing the solutions, and at regular intervals for 3600 seconds.

Proposed procedure of orthophosphate anion determination

The AM-PAT solution (6 mL), H₂SO₄ (5.5 M, 1 mL) and Na₂S₂O₃ solution (0.1192 M, 1.5 mL) were added to standard phosphate solutions (0.05 mg P mL^-1). The solution was made up to the mark with deionized water and allowed to stand for 20 minutes for maximum colour development. The absorbance was measured at 880 nm against deionized water as the reagent blank. The amount of orthophosphate (PO₄^3-) in mg P mL^-1 was determined from the absorbances of the solutions.

Method Validation

The developed sodium thiosulphate molybdenum blue method for PO₄^3- anion determination was validated according to the International Conference for Harmonisation (ICH) guidelines²⁹ under the optimized experimental conditions to determine detection limits, linearity, accuracy, precision, and recovery.

Determining detection and quantification limits

The LoD and LoQof this method were determined from standard deviation (SD) of intercepts of calibration curves obtained from replicate measurements of six blanks containing only the colourdeveloping reagents without phosphorus. Then LoD and LoQ were calculated using the equations below²⁹.

Determination of linearity of the method

The linearity of the proposed method was verified by studying absorbances of six concentrations of standard KH₂PO₄ solutions covering the range (0.01, 0.02, 0.03, 0.04, 0.05 and 0.06 mg P mL^-1). The absorption of each concentration was measured three times. The curve of absorbance against concentration was obtained by plotting and statistically by calculating the correlation coefficient of different concentrations and the blank in triplicate. The regression equation and correlation coefficient were determined.

Determination of accuracy and precision of the method

In this study, the absorption of three concentration levels (0.008, 0.02 and 0.06 mg P mL^-1) of standard KH₂PO₄ solutions within the concentration range were measured and each of them was replicated six times to evaluate accuracy and precision of the proposed methods. Precision was determined as RSD and accuracy as mean relative error (%MRE).

Determination of recovery

The accuracy of the developed method was further ascertained by carrying out recovery experiments. Deionized water was spiked with standard KH₂PO₄ solutions at two different levels that is 0.01and 0.05 mg P mL^-1 in a 50 mL volumetric flask.AM-PAT solution (6 mL), H₂SO₄ (5.5 M, 1 mL) and Na₂S₂O₃ solution (0.1192 M, 1.5 mL) were added and made up to the mark with deionized water. The absorbance of each solution and the blank was measured six times at 880 nm. The amount of phosphorus recovered was determined from calibration equations and recovery reported as RSD.

Determination of Sandell’s sensitivity

Serial dilutions of concentration range (0.01 –0.06 mg P mL^-1) were prepared from the working KH₂PO₄ standard solution and treated with AM-PAT solution (6 mL), H₂SO₄ (5.5 M, 1 mL) and Na₂S₂O₃ solution (0.1192 M, 1.5 mL). Each dilution was scanned six times at 880 nm and the absorbance was recorded. The mean absorbance at each concentration level was determined and Sandell’s sensitivity calculated using Equation 5as described by Rajendraprasad, Basavaiah, and Vinay, (2010)³⁰.

Determination of molar absorptivity

A series of phosphate concentrations in the range (0.01- 0.06 mg P mL^-1) were prepared from the working KH₂PO₄ standard solution and treated with AM-PAT solution (6 mL), H₂SO₄ (5.5 M, 1 mL) and Na₂S₂O₃ solution (0.1192 M, 1.5 mL). The absorbance of each solution was measured six times at 880 nm. Molar absorptivity was determined using Equation 6.

(where A = absorbance, Ꜫ = molar absorptivity, c = concentration of absorbing species per unit volume (mol L^-1), d = path length (cm)).

Evaluation of selectivity

Some ions and organic acids exist in water together with the orthophosphate affecting its determination. The interference due to the presence of arsenate, chloride, oxalic acid, nitrite, sulphate and copper(II) ions was also studied. In the study, salts containing interfering ionic species were dissolved in deionized water to make a 100 mg L^-1interferent solution.  The solutions of interfering species (1 mL and 5 mL) were transferred to 50 mL volumetric flasks containing 1 mL of standard KH₂PO₄ solutions (0.04 and 0.1 mg P mL^-1). To the mixtures was added AM-PAT solution (6 mL), H₂SO₄ (5.5 M, 1 mL) and Na₂S₂O₃ solution (0.1192 M, 1.5 mL) and the solutions were then made up to the mark with deionized water.  The absorbances of the resulting solutions were determined in triplicate in the presence and absence of an interferent at 880 nm against deionized water.

Application of the developed sodium thiosulphate molybdenum blue method in the determination of the orthophosphate in selected water systems

The optimized molybdenum blue method based on sodium thiosulphate as the reductant was applied in the determination of PO₄^3- in selected water systems and compared with the Murphy and Rileymethod. Water samples including distilled water and tap water collected from the laboratory together with environmental water samples collected from MakerereKikoni channel (0^o20’07.3″N32^o33’38.0″E), Kiwunya channel (0^o19’42.4″N32^o33’44.9″E) and Nanfumbambiwell (0^o19’54.8″N32^o33’46.2″E) in Kampala City – Uganda. The environmental sampling sites are located within a busy Kampala suburb where both residential and business human activities are carried out. The water samples were filtered through Whatman membrane filters (0.45 µm pore size). The first 10 mL of the filtrate of each sample was rejected and the remainder collected in labelled sample bottles for determination of PO₄^3-using the optimized method as previously described. The samples were appropriately diluted before analysis except for the distilled water and tap water samples.

Results and Discussion

Determination of absorption maxima for the phosphomolybdenum blue

The spectrophotometric method described herein involves the formation of 12-molybdophosphoric acid on condensation of the molybdate and phosphate in aqueous sulphuric acid medium followed by its reduction with sodium thiosulphate to form a phosphomolybdenum blue complex (PMB).

When the absorbance of the resultant phosphomolydenum blue (PMB) complex was scanned from 500 nm to 1100 nm, significant absorption was observed at 880 nm and 721 nm as shown in Figure 1. Therefore, the wavelength of 880 nm at which the PMB complex exhibited the highest absorbance was selected as the λ_max for the determination and was utilized in all subsequent measurements.

Figure 1: Absorption spectrum of the sodium thiosulphate reduced phosphomolybdenum blue complex formed from the solution containing 1 mL of 0.05 mg P mL-1 phosphate + AM-PAT solution (6 mL) + H2SO4 (5.5 M, 1 mL) + Na2S2O3 solution (0.119 M, 1.5 mL) diluted to 50 mL with deionized water. Click here to View figure

Optimisation of the formation and stability of the phosphomolybdenum blue complex 

The experimental conditions for theformation of PMB were optimized, by studying their effect on the intensity andstability of the final molybdenum blue colour, for the effective determination of the PO₄^3- anion in waters.

Effect of concentration of ammonium molybdate-potassium antimonyl tartrate on phosphomolybdenum blue formation

The effect of concentration of ammonium molybdate and antimonyl potassium tartrate catalyst on PMB formation was investigated with varying volumes of AM-PAT solution. A plot of absorbance of solutions treated with varying volumes of AM-PAT solution shown in Figure 2shows that the absorbance values increased with the increasing volume of AM-PAT, with maximum absorbance being achieved after addition of 6 mL of AM-PAT solution.The solution was 6.473 x 10^-3 M in ammonium molybdate and 6.1552 x 10^-4 M in antimonyl potassium tartrate. When the volume of AM-PAT was higher than 6 mL, the absorbance was found to decrease. Therefore, 6 mL of AM-PAT was used in subsequent procedures for phosphate determination.

Figure 2: Effect of different volumes of ammonium molybdate-antimonyl potassium tartrate solution + phosphate (0.05 mg P mL-1, 1mL) + H2SO4 (5.5 M, 1 mL) + Na2S2O3 solution (0.1192 M, 1 mL) diluted to 50 mL with water. Click here to View figure

Effect of acid concentrationon phosphomolybdenum blue formation

The effect of the concentration of H₂SO₄ on the absorbance of the resultant phosphomolybdenum blue was studied by using various volumes of 5.5 M H₂SO₄ in combination with the optimised volume of AM-PAT. The results of this study presented in Figure 3, show that the solution containing 1 mL of 5.5 M H₂SO₄ produced phosphomolydenum blue with the highest absorbance at 880 nm. From the graph, it can be observed that as acidity increased beyond the optimal value, the absorbance reduced. A similar observation was made by Pradhan and Pokhrel, (2013)²⁹. Therefore, a volume of 1 mL of 5.5 M H₂SO₄was used throughout the experiments for PO₄^3- determination.

Figure 3: Effect of different volumes of sulphuric acid (5.5 M, 0.25 – 2mL) + phosphate (0.05 mg P mL-1,1mL) + AM-PAT solution (6 mL) + Na2S2O3 solution (0.1192 M, 1 mL) diluted to 50 mL with water. Click here to View figure

Effect of type of acid on the absorbance of the phosphomolybdenum blue

The formation of the PMB takes place in acidic medium and it was relevant to determine the most suitable acid for the orthophosphate determination. Under this study, different acids including H₂SO_4, HCl and CH₃COOH were tested using equimolar and similar volumes of the respective acids. The results presented in Table 1 show that the highest absorbance was attained for solutions acidified with HCl followed by H₂SO₄whereas CH₃COOH exhibited the lowest absorbance. Thus, acidification of solutions for phosphate determination by the molybdenum blue method could be carried out with HCl or H₂SO₄but the former was rejected over the salt error caused by chloride interference, which results into a high standard deviation in the observed readingsas reported by Nagul et al., (2015)²⁸.

Table 1: Effect of the type of acidon the determination of the orthophosphate ion.

Entry	Type of acid	Absorbance
1	Sulphuric acid	0.195 ± 6.70%
2	Hydrochloric acid	0.206 ±17.78%
3	Acetic acid	0.011±45.05%

The value is an average of 6 replicates

Effect of concentration of reducing agenton phosphomolybdenum blue formation

The effect of Na₂S₂O₃ concentration on the formation of PMB was investigated by observing the absorbance of the PMB with varying amounts of the reductant. The absorbance of PMB increased with increasing volumes of 0.1192 M Na₂S₂O₃ with maximum absorbance obtained on adding 1.5 mL of Na₂S₂O₃ as shown in Figure 4. An increase in the amount of Na₂S₂O₃beyond the optimal volume resulted in a reduction in the absorbance of PMB. Therefore, a volume of 1.5 mL Na₂S₂O₃ was used in the subsequent experiments.

Figure 4: Effect of different volumes of Na2S2O3 solution (0.1192 M, 0.5 – 2 mL) + phosphate (0.05 mg P mL-1,1 mL) + AM-PAT solution (6 mL) + sulphuric acid (5.5 M, 1 mL) diluted to 50 mL with water. Click here to View figure

Effect of order of addition of reagents on phosphomolybdenum blue formation

In this study, the optimized volumes of the various reagents were added in different orders and taken through the proposed method. The results obtained as presented in Table 2 indicate that the change in the order of reagent addition affects absorbance values of the solutions. It was observed that the order AM-PAT + H₂SO₄ + Na₂S₂O₃ + PO₄^3- registered the highest absorbance. However, the order was not adopted because it compromised the stability of the formed PMB. Moreover, the acid can decompose the Na₂S₂O₃ as shown in Equation 7. Also, the order H₂SO₄+ Na₂S₂O₃ + PO₄^3-+ AM-PAT, exhibited a higher standard deviation in the absorbance readings. Therefore, the order PO₄^3-+ AM-PAT +H₂SO₄ + Na₂S₂O₃where the reductant is added last was followed.

S₂O₃^2- + 2H⁺ → SO₂ + S + H₂O                                         ………………… Equation 7

Table 2: Effect of order of reagents addition in the Na₂S₂O₃ molybdenum blue method

Order of addition	Absorbance±RSD%
PO43-+ AM-PAT + H2SO4+ Na2S2O3	0.148±7.97%
AM-PAT + H2SO4+ Na2S2O3+ PO43-	0.268±11.14%
H2SO4+ Na2S2O3+ PO43-+ AM-PAT	0.217±26.33%
Na2S2O3+ PO43-+ AM-PAT + H2SO4	0.113±17.74%

Value is the average of three determinations for each order

Effect of time on the formation of phosphomolybdenum blue  

The rate of formation of phosphomolybdate is time dependent (Huang and Zhang, 2008) but is also affected by a catalyst. Murphy and Riley, (1962) used potassium antimonyl tartrate as a catalyst in the single solution method of phosphate determination in sea waters. The same method was adopted with modifications to develop the Na₂S₂O₃ molybdenum blue method of orthophosphate anion determination. As seen from the graphs in Figure 5 the rate of colour development was faster in the catalyzed reaction, unlike the uncatalyzed one.The absorbance of the PMB increased steadily up to 20 min after which it increased slightly with time for 60 min studied.Thus, the optimal time lag after theaddition of the reductant to thereading of the absorbance of the formed molybdenum blue complex was 20 minutes. This implies that the rate of molybdenum blue colour formation is slightly higher with ascorbic acid reductant compared to sodium thiosulphate, since the former requires 10 minutes to attain optimal colour development.

Figure 5: Effect of time on the colour stability of Na2S2O3 reduced phosphomolybdate solution Click here to View figure

Validation of the sodium thiosulphate molybdenum blue method for orthophosphate determination

Calibration curve and sensitivity

The calibration curve for orthophosphate analysis obtained by plotting absorbance as a function of the concentration of orthophosphate (in mg P mL^-1) at 880 nm is shown in Figure 6. The plot is linear in the range of 0.005 – 0.06 mg P mL^-1. The amount of orthophosphate within this range can be determined directly and for those above this range, the sample should be diluted. The molar absorptivity and Sandell’s sensitivity determined from the calibration curve at 880 nm were 57526 L mol^-1 cm^-1 and 0.2835 µg cm^-2 respectively. The values were found to be within acceptable limits. The LoD and LoQ obtained using the proposed method in comparison to those achieved with the Murphy and Riley methods are presented in Table 3. The results show that the present method based on the sodium thiosulphatereductant offers a lower detection (LoD) and quantification (LoQ) limits compared to the commonly used Murphy and Riley method. This suggests that the sodium thiosulphate method can be used to determine low levels of P in water systems.

Figure 6: Calibration curve for the determination of orthophosphate Click here to View figure

Table 3: Comparison of detection limits obtained with the proposed method and the Murphy and Riley methods of orthophosphate determination

Method	LoD (mg P mL-1)	LoQ (mg P mL-1)
Murphy and Riley	8.31×10-3	0.0252
Present	2.213×10-3	5.538×10-³

n=6 (average of six readings); LoD and LoQ values of the Murphy and Riley method were calculated from experimentally determined values.

Accuracy and Precision

The precision of the method was determined by repeat analysis of samples at different concentrations and results were expressed as standard deviation (SD) and relative standard deviation (%RSD). Results of the precision studies for 6 replicate determinations of different orthophosphate levels exhibited %RSD values in the range 1.72 to 7.01 % (Table 4). The RSD values of less than 20% are regarded as good indicating the method’s repeatability when used for analysing orthophosphate levels in waters. The results obtained for replicate determinations of 0.008, 0.02 and 0.06 mg P mL^-1 phosphate were analysed by ANOVA. The F-critical 4.414 was less than the F-calculated 32.91, 32.77 and 32.29 for the respective concentrations. An indication that the results of the orthophosphate determination obtained using the present sodium thiosulphate molybdenum blue method are precise.

Table 4: Evaluation of precision of the Na₂S₂O₃ molybdenum blue method.

Added concentration (mg P mL-1)	Detected concentration (mg P mL-1)	MRE (%)	SD	RSD (%)
0.008	0.0079	1.53	1.44 x 10-4	1.84
0.02	0.0187	6.75	1.31 x 10-3	7.01
0.06	0.0584	2.64	1.00 x 10-3	1.72

n=6 (average of ten readings), MRE is mean relative error

In this study, the accuracy of the developed method was validated by calculating the mean percent recovery of spiked samples for six replicates at different concentration levels prepared from independent stock solutions. Recoveries above 90% were registered for the different concentration levels. The RSD values were within the acceptable range of less than 20% (Table 5). Therefore, this ably demonstrates that the present sodium thiosulphate molybdenum blue method is suitable for the determination of low levels of the orthophosphate and the results generated are accurate.

Table 5: Evaluation of accuracy of the Na₂S₂O₃ molybdenum blue method.

Added concentration (mg P mL-1)	Recovery ±RSD%
0.008	97.68±2.88
0.02	97.5±10.12
0.06	98.67±2.56

n = 6 (average of six readings)

Effect of interfering substances

The effect of interfering cations, anions and organic acids on phosphate determination using the developed sodium thiosulphate molybdenum blue method was also examined. In this study, ions that are known to affect the determination PO₄^3- in water samples using the molybdenum blue methods were added to standardsolutions and analysed using the present method. The results show that interferents affected the analysis differently.

The interference caused by arsenate ion is shown in Figure 7. The absorbance of molybdenum blue solutions formed increased in presence of AsO₄^3-(Figure 7b). The AsO₄^3- ion has a tetrahedral geometry like the orthophosphate and can undergo a similar reaction with molybdate forming heteropolyacids. Upon reduction, they form arsenomolybdenum blue species with an absorption maximum close to that of the phosphomolybdenum blue. Indeed, a previous study utilized the molybdenum blue method to determine AsO₄^3-in natural water samples at 865 nm as shown in Figure 7a³², which is likely to affect orthophosphate determination at 880 nm. When AsO₄^3-ions were introduced into in the reaction mixture, they interfered with phosphorus analysis causing a shift in the λmax. Investigations carried out at two orthophosphate levels (0.04 and 0.1 mg P mL^-1) show that absorbance increased with increase in AsO₄^3-concentrations. Therefore, AsO₄^3-has positive interference on PO₄^3- determination. In this respect, in the analysis of water samples containing significant amounts of the arsenate using the present sodium thiosulphate molybdenum blue method, this interferent must be removed using standard procedures before determination of the orthophosphate.In contrast, Cl^– ions exhibitednegative interference in the orthophosphate determination possibly by decreasing the analytical signal sinceCl^– disrupts the formation of PMB²⁸.

Figure 7: Absorbance spectrum of arsenate ions (a), Effect of arsenate ions on molybdenum blue formation (b). Click here to View figure

Results of the investigation for the effect of oxalic acid on molybdenum blue formation are shown in Figure 8. The results show a reduction in absorbance of PMB complex with increase in the oxalic acid concentrations. Generally, organic acids interfere inphosphomolybdenum blue methods by sequestering Mo(VI) and destroying 12-molybdophosphoric acid. This is possible because organic acids like oxalic acid coordinate in a bidentate manner with Mo(VI) to form a stable six membered complex³³. This results in a reduction in the absorbance of the PMB. 

Figure 8: Effect of oxalic acid on molybdenum blue formation Click here to View figure

The effect of the nitrite ion on the molybdenum blue formation is shown in Figure 9. Presence of the nitrite ion decreases the absorbance of the reduced phosphomolybdate complex. The nitrite ion is an oxidizing agent, thus, it oxidises molybdenum blue according to Equation 8 which is followed by a reduction in absorbance. Similar observations were made in the determination of nitrate and nitrite in water, meat products and vegetables using phosphomolybdenum blue method; a reduction in the intensity of the blue colour was proportional to nitrite content³⁴.

[PMo^V₄Mo^VI₈O₄₀]^7- + NO₂^– → [PMo^VI₁₂O₄₀]^3- + NO  ………………….. Equation 8

Figure 9: Effect of nitrite on molybdenum blue formation Click here to View figure

Results of the effect of copper(II) ions on molybdenum blue formation presented in Figure 10, show that the absorbance of the complex decreased with increasing concentrations of Cu²⁺ ions added to PO₄^3- solutions. The reduced absorbance could be a result of inhibition of Mo(VI) reduction which is caused by Cu²⁺ ions^35,36. Like Cu²⁺, Pb²⁺ ions also inhibit Mo(VI) reduction leading to reduced intensity of molybdenum blue formed³⁴.

Figure 10: Interference of Cu2+ ion on molybdenum blue formation Click here to View figure

Determination of levels of orthophosphate anion in selected water systems

The developed sodium thiosulphate molybdenum blue method was applied in the determination of the orthophosphate in selected natural waters and the results are presented in Table 6. The results show that the PO₄^3- was present in the different water samples in varying concentrations.Samples from MakerereKikoni Channel and Kiwunya Channel exhibited higher levels of P of 0.03 ppm and 0.028 ppm respectively. The high PO₄^3-concentration in water from these channels could be attributed to the various sources of phosphorus such as solid and liquid wastes generated from households, shops, restaurants and kiosks along the channel. Also, the animal excreta and pit latrines located in the vicinity discharge into the channel especially on rainy days. Moreover, there are several washing bays where youth use detergents some of which are P based, to wash motorcycles and bicycles. Further on, the elevated P levels could also be due to natural decomposition of rocks and minerals, weathering of soluble inorganic materials and leaching of biomass³⁷. On the other hand, the amount of orthophosphate in the distilled water and tap water samples were below detection limits.

As shown in Table 6, the results obtained forPO₄^3- determination in water samples using the present sodium thiosulphate method were compared to those obtained using the Murphy and Riley, 1962 method that employs ascorbic acid reducing agent. The results obtained for the different samples using the two methods are in agreement.  The correlation of the two methods was found to be 0.996 for determination of PO₄^3-in five water samples. This demonstrates the applicability of the developed method for the accurate determination of the orthophosphate in environmental water samples.

Table 6: Determination of phosphate in water samples.

Sample	Phosphate concentration ± SD (mg P mL-1)
	Ascorbic acid method	Na2S2O3 method
Distilled water	ND	ND
Tap water	ND	ND
MakerereKikoni channel water	0.0336 ±0.001	0.0300 ±0.05
Kiwunya channel water	0.0316 ±0.0006	0.0278 ±0.005
Nanfumbambi well water	0.016 ±0.001	0.0177 ±0.009

Results based on five measurements. ND = detected. SD = standard deviation

Conclusion

A simple and rapid spectrophotometric method for the determination of low levels of orthophosphate ions in water is reported. The method involves thereduction of phosphomolybdate complex formed by condensation of the molybdate and phosphate in anaqueous acid medium using Na₂S₂O₃ solution. The result is theformation of molybdenum blue which exhibited a maximum absorption at 880 nm. The method is simple and cheap compared to other methods since it does not require sophisticated instruments or involve extraction of the analyte and the Na₂S₂O₃ reducing agent employed to reduce phosphomolybdate is a readily available laboratory chemical. The optimized method was successful employed in the determination of PO₄^3- in water samples and the results of the analysis compared favourably with the Murphy and Riley, 1962 ascorbic acid method. Also, the present sodium thiosulphate molybdenum blue method exhibited low LoD and LoQ in the determination of the orthophosphate compared to the ascorbic acid molybdenum blue which is the most commonly used method for this analysis.

Acknowledgement

The authors would like to acknowledge the Organisation for the Prohibition of Chemical Weapons (OPCW), the African-German Network of Excellence in Science (AGNES) and the German Academic Exchange Service (DAAD) for funding this study. The authors would also like to thank the International Science Programme (ISP), Uppsala University, Sweden, for funding some of the equipment used in the study. Support by the School of Chemistry and Physics, University of KwaZulu Natal – Westville, Departments of Chemistry, Mbarara University of Science and Technology and Department of Chemistry, Makerere University is also gratefully acknowledged.

Conflict of Interest

The authors declare that they have no conflict of interest.

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