A Simple Spectrophotometric Determination of Phosphate in Sugarcane Juices , Water and Detergent Samples

A simple spectrophotometric method is developed for the determination of phosphate in sugar cane juice, water and detergent samples. The method is based on the formation of phosphomolybdate with added molybdate followed by its reduction with sodium sulphide in aqueous sulfuric acid medium. The system obeys Lambert-Beer’s law at 715 nm in the concentration range 0.3-12.24 ppm. Molar absorptivity, correlation coefficient and Sandell’s sensitivity values were found to be 6.1x10 mol cm, 0.999 and 0.0156 μg cm respectively. The results obtained were reproducible with acceptable standard deviation 3.7% and relative error 3.4%. For a comparison of the method phosphate present in various samples were also determined separately following an official method. The results of the developed method compare well with those of the official method.


Introduction
Phosphorus is the eleventh most abundant element on the surface of the earth and is most commonly found as phosphate.It plays an important role in biochemical processes and is a key factor in the eutrophication of surface water 1 Increased phosphate concentrations are linked with increasing rates of plant growth 2 .The analytical chemistry of phosphorus is very important in many fields, for example, medical and clinical science, agriculture, metallurgy and environmental science 3 .Moreover, in recent years large quantities of phosphate have been used in beverages 4 , detergents 5 , fertilizers 6 and also in sugar industries [7][8][9] .The literature [7][8][9] gives the importance of phosphate during the sugarcane juice clarification leading to cane sugar manufacture.In this context, the chemical action of phosphate during clarification of sugarcane juice assumes significant role particularly when the juice is deficient in natural phosphate content in the range of 300-350 ppm as P 2 O 5 which is essential for good clarification.In case of less phosphate content in sugarcane juice, soluble phosphate addition is the only alternative to achieve the target value by adding orthophosphoric acid, single super phosphate or triple super phosphate.Therefore, it becomes very important to determine the amount of phosphate in sugarcane juice.As a consequence, various phosphate determination procedures have been reported [10][11][12][13][14][15][16][17][18][19][20][21][22] which include for example, titrometry 10 , complexogravimetry 11 , colorimetry 12,13 , atomic absorption spectroscopy, 14 flow injection analysis 15 , HPLC 16 and spectrophotometry methods [17][18][19][20][21][22] .Among such methods spectrophotometry involving molybdovanadate 23 and ammonium molybdate 16 are most commonly used 16 .In ammonium molybdate spectrophotometric method, different reductants have been employed such as tin(II) chloride 23,25 .ascorbic acid 26 and 1-amino-2naphthol-4-sulfonic acid 27 , Some of these methods also involve complicated and expensive equipments 16 and need extraction procedure 20,23 and such techniques are usually not available in common laboratories.Though there are large number of methods [10][11][12][13][14][15][16][17][18][19][20][21][22] available for the determination of phosphate but such methods used in sugar cane juice for phosphate determination are unread, instead only a few methods 8,9,23,25 have been used.Therefore, it becomes very important [7][8] to determine the phosphate in sugar cane juice 7 and sodium sulphide is found to be a very effective reducing agent 28 but not commonly used for phosphate determination. 28Considering the importance of phosphate and its determination in sugar cane juice an attempt is made here to develop the simple spectrophotometric method which is based on the formation of phosphomolybdate with added molybdate 16,24 followed by its reduction with sodium sulphide 28 in aqueous acidic medium.

Experimental
Elico spectrophotometer model SL 171 (Hyderabad, India) with 1 cm matched quartz cells, Sartorius digital balance readable 0.0001g were used.

Reagents
All the chemical reagents used were of analytical grade and the water used was distilled water.Weighed amount of ammonium molybdate, 1.7081 g was dissolved in about 150 mL of warm water 27 ; slightly milky solution obtained was cooled to room temperature.It was then transferred into a 250 mL volumetric flask and diluted to the mark with water.0.05g of sodium sulphide was transferred into a clean 100 mL beaker.It was dissolved in about 50 mL of water and then the solution was transferred into a 100 mL volumetric flask.The beaker was washed 3-4 times with water and washings were also transferred into the flask and the solution was diluted to the mark with water.The solution was standardized iodometrically 28 before use.0.1145 g of disodium hydrogen phosphate was transferred into a 150 mL beaker 29 .It was dissolved in water, then; the solution was transferred into a 250 mL volumetric flask and diluted to mark with water.The working solutions were prepared by diluting 5 mL of this solution to 50 mL with water.

Recommended procedure
A series of 10 mL volumetric flasks were arranged.To each flask 0.5 mL of 0.387M ammonium molybdate, 3mL of 0.25N sulfuric acid and aliquots of disodium hydrogen phosphate corresponding to 0.3-12.24ppm (0.1, .2,0.3, 0.5, 1.0 , 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 mL) were added.Finally, to each flask, 1 mL of 2.0833x10 -3 M sodium sulphide solution was added.Then, each solution was let at room temperature for about 20 min.The absorbances of solutions were measured at 715 nm against water.

Sugarcane juice sample preparation
Sugarcanes were collected from the sugar cane fields situated around Mysore.Sugar cane juice was extracted from such sugar canes by laboratory crusher 8 ; the obtained juice was collected and stored in a separate reagent bottle.The same juice of 5 mL of that was diluted to 100 mL with water and filtered to separate any suspended particles 25 .

Detergent sample preparation 5
5g washing powder of Indian origin was placed in an oven for about half an hour at about 350 0 C, the obtained ash was taken in a 150mL beaker.It was dissolved in about 50 mL water and the solution was made acidic by adding dilute sulfuric acid.It was heated on water bath for about 10 min.to expel the hydrogen sulphide, nitrite etc.It was filtered and the filtrate was transferred into a 100 mL volumetric flask and diluted to the mark with water, later it was used for its phosphate analysis.
Water sample 2,3 Water sample was collected from Kukkarahalli Lake which is located in Mysore city.Sample was filtered through a cellulose membrane filter, the filtrate containing orthophosphate, condensed phosphate (Pyro, Meta and poly phosphate) and organically bound phosphorous, all of which may be present in soluble form and in suspension.As is well known that only orthophosphate can be determined directly by phosphomolybdate procedures, pyro, tripoly and poly phosphate are completely hydrolyzed to orthophosphate by acidification and heating (30 min.)and it was used for its phosphate analysis.

Results and Discussion
The developed method is based on the formation of phosphomolybdate 11,17,23,25 due to the reaction between molybdate and phosphate followed by its reduction with sodium sulphide in aqueous sulfuric acid medium.Under optimized experimental condition, with fixed concentration of molybdate and reducing agent , the colour intensity was found to be proportional to the amount of phosphate present in disodium hydrogen phosphate, sugarcane juices, water and detergent samples.The reaction conditions as well as the various experimental parameters affecting the development and stability of the coloured complex were carefully investigated and optimized for quantitative determination of phosphate in various samples analyzed.The experimental variables such as concentration of ammonium molybdate, concentration of sulfuric acid, concentration of sodium sulphide, order of addition of the reagents and also colour stability of the complex were optimized for the effective determination of phosphate.The stability constant and composition of the system were also determined and values were found to be 1.992x10 6 L mol -1 and 1:1 with respect to molybdate and phosphate.For a comparison of the results, phosphate present with those various samples was also determined separately following the official method 25 .The results obtained for phosphate both by the proposed and official method 25 are shown in Table 1and 2 which account for the complimenting nature of the results from both the methods.Therefore, the proposed method could be either a substitute to an official method 25 or it could also be an independent method for the determination phosphate in the sugarcane juice samples.

Optimization Effect of concentration of ammonium molybdate
First 5.528x10 -3 M ammonium molybdate solution was prepared 29,31 .Three series, each series having three labeled 10 mL volumetric flasks were arranged .To each flask of the first series 0.1 mL of 5.528x10 -3 M molybdate solution was added.To each flask of the second series 0.3 mL of 5.528x10 -3 M molybdate solution was added.To each flask of the third series 0.5 mL of 5.528x10 -3 M molybdate solution was added.To the flasks of each series, measured but various volumes (0.2, 0.5, 1.0 mL) of phosphate (30 ppm) solution, 1 mL of 2.0833x10 -3 M sodium sulphide solutions were added.All the solutions were diluted to the mark with water.The absorbance of the solutions were measured at 715 nm .The first series solutions containing 0.1 mL of molybdate showed absorbance 0.01, 0.02, and 0.05 for 0.2, 0.5,1.0mL of phosphate.It yielded a straight line, but ran almost parallel to x-axis, indicating low sensitivity.A second series of solutions containing 0.3 mL of molybdate solutions showed absorbance value 0.02, 0.03and 0.05 for 0.2, 0.5and 1.0 mL of phosphate respectively.This also yielded a straight line but indicated low sensitivity when the absorbance is plotted against concentration of phosphate.A third series of solutions containing 0.5 mL of ammonium molybdate solutions showed absorbance values of 0.07, 0.09, and 0.15 for 0.2, 0.5 and 1.0 mL of phosphate respectively .The absorbance values were found to increase with concentration of phosphate and the colour of the solutions were stable up to 2h.Therefore, 0.5 mL of ammonium molybdate was more preferred than its volume used in the earlier two sets solutions for the determination of phosphate.

Effect of concentration of sulfuric acid
The effect of concentration of sulfuric acid on absorbance was investigated to achieve high absorbance via taking various volumes (1-3 mL) of 0.25 N of sulfuric acid ,0.5 mL of ammonium molybdate , 1 mL of phosphate (30 ppm) and 1 mL of 2.0833x10 3 M sodium sulphide solutions were added to the 10 mL volumetric flasks.Then, the solutions were diluted to the mark with water.Absorbance of each solution was measured at 715 nm.The absorbance of the solutions were 0.02, 0.05and 0.15 respectively for 1,2 and 3 mL of 0.25 N sulfuric acid .The absorbance values indicated that the solution containing 3 mL of 0.25 N sulfuric acid appeared to be more sensitive.Therefore, it was used throughout the experiment to determine the phosphate.

Effect of concentration of sodium sulphide
The concentration influence of reducing agent was studied as above with different volumes (0.2, 0.5, 0.7, 1.0mL) of 2.0833x10 -3 M of sodium sulphide.The absorbance values were found to be 0.05, 0.1 and 0.16 respectively for sodium sulphide.Based on these results; 1.0 mL of this reagent was selected for the construction of calibration graph.

Effect of order of addition of the reagents
Different orders of the reagent addition were studied using optimised amounts of reagents following the recommended procedure .The results obtained have shown that the order of reagents addition do not effect absorbance values.But for maintaining the uniformity of the order of reagents addition as mentioned in the recommended procedure was followed throughout course of the determination of phosphate.

Calibration Graph
Under the optimum condition, a good linear relationship Figure 1 was found to exist between the absorbance of the system and concentration of phosphate 0.3-12.24ppm with a straight line having slope 0.0336(Cal.)and intercept 0.4667.Molarabsotptivity, correlation coefficient, and Sandell's sensitivity values were calculated and were found to be 6.103x10 3 mol -1 cm, -1 0.999 and 0.0156 µg cm -2 respectively.The precision and accuracy of the method were studied by analyzing a series of solution containing known amount of phosphate (0.3, 3.0 and 10.7 ppm) by using recommended volumes and concentration of the reagents.The precision of the method as expressed by relative standard deviation was less than 3.7% whereas the accuracy expressed by the calculated relative error was 3.4%.

Effect of foreign ions
The effect of foreign ions were examined by using 3 ppm of phosphate and the results obtained are shown in Table 3.Most of the cations and anions commonly found in sugar cane juice 7,8 do not interfere but iron; copper and zirconium interfere at 20, 12, and 11 ppm respectively.

Conclusions
,25,27,32 .(i) It is more sensitive when compared to the reported method 17 .(ii) The preparation of reducing agent in this method is very simple when compared to that in the official method 25 .(iii) It neither involves extraction 19 nor sophisticated instruments like HPLC, 16 flow injection spectrophotometer 15 and atomic spectroscopy 14 , nor requires strict control of pH 23,27,32 or temperature or heat treatment 32 .As the method is observed to be working satisfactorily for the determination of phosphate in various samples giving the results which are not only comparable with the results of phosphate determined separately from an official method 25 and also reproducible as revealed by the values of statistical parameters like standard deviation, 3.7% and relative error, 3.4%.Therefore, the method could be employed for the determination of phosphate as an independent or a complimentary one to the official method 25 .

Figure 1 .
Figure 1.Calibration graph for the determination phosphate under optimized experimental condition.

Table 1 .
Determination of phosphate in sugar cane juices.

Table 2 .
Determination of phosphate in detergent powders

Table 3 .
Effect of foreign ions on the determination of phosphate.