ABSTRACT The study of this report was to analyse phosphate in a solution

ABSTRACT
The study of this report was to analyse phosphate in a solution. The main objective was to determine the concentration of phosphate in an unknown solution. The level of phosphate in the sample solution was evaluated by molybdenum phosphorus blue method. A spectrophotometer was used with which the absorbance of the solutions provided were read at a particular wavelength of 880 nm where maximum absorption of light occurs during the phosphate test. A graph of absorbance against concentration of the solution is plotted. Using Beer’s Law: (A= ?lc) and line of best fit: (y=0.3726x) , the concentration of phosphate in five different unknown solutions has been found knowing their absorbance at 880 nm wavelength. The concentration of phosphate in the unknown solutions (A, B, C, D, E) was displayed and it should be noted that A, B and E had high concentration of phosphate of 5.77, 2.66 and 2.02 mg/L respectively compared to C (0.757 mg/L) and D (0.773 mg/L). If these high amounts of phosphate were found in water surfaces, this would be a threat to aquatic life and the environment in general. This is why it is important to perform this experiment to analyse the quantity of phosphate in water so that appropriate measures can be taken.
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TABLE OF CONTENTS

ABSTRACT 2
LIST OF TABLES 4
LIST OF FIGURES 4
LIST OF EQUATIONS 4
1. INTRODUCTION 5
2. AIMS AND ODJECTIVES 6
3. LITERATURE REVIEW 7
3.1 Importance of phosphorus 7
3.2 Types of phosphates 7
3.3 Sources and threat of phosphates in water 7
3.5 How to determine concentration of phosphate in water 8
3.6 What is spectrophotometry? 8
3.7 Application of Beer’s Law 9
4. METHODOLOGY 10
4.1 Apparatus used 10
4.2 Chemicals used 11
4.3 Procedure 11
4.4 Determination of phosphate in the unknown solution 12
4.5 Dangers and safety measures 12
6. CALCULATIONS 14
6.1 Using Beer’s Law equation 14
6.2 Error analysis 14
7. DISCUSSION OF RESULTS 15
7.1 Colour formation 15
7.2 Plotting the graph 15
7.3 Values obtained in the experiment 16
8. CONCLUSION 16
9. REFERENCES 17
10. APPENDICES 18

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LIST OF TABLES

Table 1: List of reagents, dangers and safety measures 12
Table 2: Absorbance of different concentration of the sample solutions 13
Table 3: Absorbance of the unknown solution 13
Table 4: Concentration of phosphate in the unknown solution 14

LIST OF FIGURES
Figure 1: Diagram of the main components in a spectrophotometer 8
Figure 2: Diagram of a beam of light as it passes through the solution vial 9
Figure 3: Hach DR 2500 spectrophotometer 10
Figure 4: PhosVer? 3 phosphate reagent powder pillow 11
Figure 5: Graph of Absorbance against Concentration of phosphate 13
Figure 6: Results on completion of the chemical reaction 15

LIST OF EQUATIONS
Equation 1 : First step reaction to determine presence of orthophosphate 5
Equation 2 : Second step reaction to determine presence of orthophosphate 5
Equation 3: Beer’s Law Equation 6
Equation 4: Transmittance 9
Equation 5: Absorbance 9
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1. INTRODUCTION
Quantitative analysis demonstrates what amount of one or more substances are present in a sample. (Bhutia T.K., 2016). One type of quantitative analysis in chemistry is the determination of phosphate in a sample solution. The most stable form of phosphate in water is orthophosphate which is also mentioned as reactive phosphorus. (Murphy S., 2007). The method used to ascertain the amount of phosphate ions (PO43-) in the given sample solution was the PhosVer? 3 (Ascorbic Acid) Method 8048. In another term, it is also called the spectrophotometric molybdenum blue method.
In this experiment ammonium molybdate has reacted with antimony potassium tartrate in potassium pyrosulfate (acid medium) along with phosphate diluted solutions. A complex named antimony-phospho-molybdate was formed which is yellow in colour. In the presence of ascorbic acid (chemical name for Vitamin C), the complex was immediately reduced to an intensely molybdenum blue complex. (Murphy and Riley, 1977 cited Doolittle P., 2014). Since the reduction reaction has instantly taken place the yellow colour was not observed. All the listed components for the two step reactions were present in the PhosVer 3 Phosphate Reagent except the diluted solutions of orthophosphate.
Ammonium
Molybdate
(NH4)6Mo7O24.4H2O) + Antimony
Potassium
Tartrate
(K(SbO)C4H4O6.?(1/2) H2O) + Potassium
Pyrosulfate
(K2O7S2) + Orthophosphate
(PO43-)

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Antimony-phospho-molybdate complex
(Yellow Colour)
Equation 1 : First step reaction to determine presence of orthophosphate
Antimony-phospho-molybdate
(Yellow Colour) + Ascorbic acid
(C6H8O6)
(Vitamin C) ? Molybdenum complex
(Mo)
(Blue Colour)
Equation 2 : Second step reaction to determine presence of orthophosphate

In this experiment Beer’s Law equation short for Beer-Lambert Law equation was used to find the concentration of phosphate ions in the sample solution using a spectrophotometer. This law states that the concentration of a chemical substance in that case orthophosphate, is directly proportional to the absorbance of light through the sample solution (sample containing orthophosphate). (Helmenstine A.M., 2017). The Beer’s Law is represented as shown below:
A= ?cl
Equation 3: Beer’s Law Equation
A: Absorbance of light (no units)
?: Molar absorptivity (mol-1 m-1)
l: Path length of vial used (m)
c: Concentration of substance (orthophosphate) in solution (mol L-1 / mol dm-3)

In this experiment concentrations of PO43- ions ranging from 0.50 to 2.50 mg/L by interval of 0.5 were used. This resulted into five different concentrations of phosphate ions which were used in the test. Using the spectrophotometer, program 490 P React. PP was selected which is a specific program for the determination of phosphate. (Chemistry For Engineers Practical Handout). A wavelength of 880 nm was programmed into the spectrophotometer machine as at this particular wavelength, maximum absorption of light occurs when testing for phosphate. (Douglas R., 2007)

2. AIMS AND ODJECTIVES
To understand the principle of a spectrophotometric analysis using a spectrophotometer
To determine the concentration of phosphate in an unknown solution using Beer’s Law
To determine amount of phosphate in water samples from rivers, lakes, reservoirs to ensure the water is safe for consumption

3. LITERATURE REVIEW
3.1 Importance of phosphorus
Phosphorus is an important factor in the growth of plants on land as well as in water. In enough amount of phosphorus, plants can develop immunity against diseases hence grow well. This can be shown by hydroponic gardening where the amount of phosphorus can be controlled resulting in more healthy plants. (Tajer A., 2016). In nature phosphorus is found in the form of phosphate ions (PO43-).
Phosphorus in any form in water is relatively low in concentration as the aquatic plants and animals are very keen to intake this nutrient. (Abbasi S.A., 1998). Here are a few statistical values of amount of phosphate: 0.001 mg/l in pure water, 0.005 to 0.020 mg/l in natural surface water, 0.020 mg/l in groundwater and up to 200 mg/l in seawater. (Abbasi S.A., 1998).
3.2 Types of phosphates
Inorganic phosphates include orthophosphates and polyphosphates
Orthophosphates (reactive phosphorus)
Polyphosphates (condensed phosphates) are unstable in water and are directly converted to orthophosphate
Organic phosphates are phosphates found in living organisms
3.3 Sources and threat of phosphates in water
In nature phosphates reach water surfaces from minerals and rocks during erosion. A large amount of phosphate comes from human activities such household and industrial sewage, fertilisation and detergents. (Green J., 2017).
In a situation where the amount of phosphorus in water is so high and that plants and animals cannot intake all of the nutrient, eutrophication can occur. (Abbasi S.A., 1998). Eutrophication also known as the effect of algal blooms is a major concern. Excessive amount of phosphates in water increases the growth of algae. Bacteria decompose the dying algae and take in the dissolved oxygen in water. As a result the aquatic plants and animals suffocate and die due to lack of oxygen. (Green J., 2017)
Therefore the need of performing an orthophosphate test of water sample from water courses is crucial to know how alarming the situation is and to take appropriate measures at an early stage.
3.5 How to determine concentration of phosphate in water
There are many different tests to analyse the amount of phosphate in water namely: orthophosphate test, acid hydrolysable phosphate test and the total phosphorous test. Among the types of phosphates listed above only orthophosphate can be immediately analysed. (Dabkowski B. and White M., 2016). In this experiment the orthophosphate test involving the ascorbic acid and spectrophotometric molybdenum blue method was used.
3.6 What is spectrophotometry?
Spectrophotometry is a process where the concentration of a substance in an unknown sample can be found by measuring the absorbance of light of that substance in the solution. (Douglas R., 2007). The device used for this experiment was the Hach DR 2500 spectrophotometer having a wavelength range of 365 to 880 nm.
In figure 1 below, the process that happens inside the spectrophotometer for the quantitative analysis of phosphate is shown. A white source of light (light having more than one wavelength) usually produced by a tungsten lamp was made to pass through a monochromator. (Douglas R., 2007). A prism inside the monochromator has dispersed the light and by moving the exit slit, only one wavelength of 880 nm left the monochromator. (Heda N., 2013).

Figure 1: Diagram of the main components in a spectrophotometer
(http://namrataheda.blogspot.com/2013/07/spectrophotometry-part-2-uv-visible.html)
The single beam of light was then passed through a 10 ml vial (cuvette) containing the blue solution of molybdenum. The solution had absorbed some light and the remaining light was transmitted as shown in figure 2 below. The amount of light absorbed depends on the length of the vial and concentration of the solution. The transmittance and absorbance of light are found using the following equations:

T=It/Io
Equation 4: Transmittance

A= -log?(T)= -log?(It/Io)
Equation 5: Absorbance

Io: Intensity of light that entered the solution (Wm-2)
It: Intensity of light that left the solution (Wm-2)
l: path length of vial (m)
T: Transmittance of light (no units)
A: Absorbance of light (no units)

Figure 2: Diagram of a beam of light as it passes through the solution vial
(http://www.thefullwiki.org/General_Astronomy/Molecular_Emission_and_Absorption)
Spectrophotometry is used for the quantitative analysis of phosphate because the process requires neither sophisticated instruments nor extraction compared to the other methods. (Adelowo F.E. et al, 2016)

3.7 Application of Beer’s Law
The mathematical expression of Beer’s Law A= ?lc states that the absorbance is directly proportional to the concentration of the substance. It demonstrates that the concentration of the substance in a solution is also proportional to the colour intensity of the solution. In simple terms, if during the test a darker blue colour was obtained, it means that the solution contained a large amount of phosphate. (Douglas R., 2007).

4. METHODOLOGY
In this experiment 50mg/L of phosphate standard solution was prepared. Five separate dilutions of the given solution were made thereby obtaining five different concentration of phosphate. The powder reagent provided was mixed with each of the diluted solutions separately. The absorbance of light of each mixed solutions was noted and a graph of absorbance against concentration of phosphate was plotted. From the graph the concentration of phosphate in an unknown solution was determined using the Beer’s Law equation.
4.1 Apparatus used
Hach DR 2500 spectrophotometer
It was used to measure the absorbance of light of the diluted phosphate solutions and the unknown solution at a wavelength of 880 nm.

Figure 3: Hach DR 2500 spectrophotometer
(http://www.balmann.co.kr/Details/Product/Analysis-DR2500(HACH).asp)
50 ml Beaker
The standard phosphate solution was poured into the beaker for the solution to be pipetted easily.

15 ml Graduated Pipette
It was used to transfer volumes of the phosphate standard solution into five different volumetric flasks.

100 ml Volumetric Flasks
Five different volumes of the standard phosphate solution ranging from 5 ml to 25 ml were transferred into the volumetric flasks and distilled water was added up to the 100 ml marks obtaining at last five diluted solutions of different concentration.

10 ml Pipette
It was used to transfer 10 ml of each diluted solution into 10 ml vials into which the phosphate reagent was added (sample solutions). It was also used to transfer 10 ml of the diluted solution into one 10 ml vial without adding the phosphate reagent (blank sample) as a reference. At last it was used to transfer 10 ml of the unknown solution into a 10 ml vial.

10 ml vials
They were used to store the sample solutions, the blank solution and the unknown solution. They were then placed each at a time in the spectrophotometer to measure the absorbance.

Timer
It was used to set a 21/2 minute reaction (shaking for 30 seconds and 2 minutes for the reaction)
4.2 Chemicals used
Phosphate standard solution, 50 mg/L
Distilled water
PhosVer? 3 phosphate reagent powder pillow 10 ml

Figure 4: PhosVer? 3 phosphate reagent powder pillow
(https://www.hach.com/phosver-3-phosphate-reagent-powder-pillows-10-ml-pk-100/product?id=7640196043)
4.3 Procedure
Program 490 P React. PP was started and a wavelength of 880 nm was selected.
15 ml of the phosphate standard solution was transferred into a 100 ml volumetric flask.
Distilled water was added to the volumetric flask up to the 100ml mark.
The contents were mixed by carefully inverting the volumetric flask.
The sample solution was obtained by transferring 10 ml of the diluted solution into a 10 ml vial.
A PhosVer? 3 phosphate reagent powder pillow was cut using scissors and all its contents were added to the prepared sample solution vial.
The vial was instantly closed and shaken vigorously for 30 seconds.
A further 2 minute reaction was set using a timer.
Meanwhile the blank sample was obtained by transferring 10 ml of the diluted solution into another 10 ml vial.
Using tissue paper, the vial containing the blank sample was cleaned (removing traces of fingerprints) and was inserted into the cell holder of the spectrophotometer.
The command ZERO was selected to zero the spectrophotometer.
When the timer was beeped, step 10 was repeated for the sample solution, the command READ was selected and the absorbance was noted.
The experiment was repeated for different volumes of the phosphate standard solution hence different concentration and each absorbance was noted.
A graph of absorbance against concentration of the phosphate solutions was plotted.
4.4 Determination of phosphate in the unknown solution
10 ml of the unknown solution was transferred into a 10 ml vial.
The blank sample that was prepared before was reinserted into the cell holder to zero the spectrophotometer.
The unknown sample was then placed into the cell holder of the spectrophotometer, command READ was selected and absorbance was noted.
4.5 Dangers and safety measures
Reagent Dangers Safety measures
Phosphate standard solution Causes eye irritation and skin irritation in case of contact Wear eye goggles and gloves
PhosVer? 3 phosphate reagent Causes eye irritation and skin irritation in case of contact
Causes respiratory tract irritation if accidentally inhaled Wear eye goggles and gloves
Wear mask when adding the powder
Table 1: List of reagents, dangers and safety measures
5. TABLE OF RESULTS
Vials 1 2 3 4 5
Concentration (mg/L) 0.5 1.0 1.5 2.0 2.5
Volume of solution to be diluted (ml) 5 10 15 20 25
Absorbance 0.192 0.538 0.672 0.752 0.791
Table 2: Absorbance of different concentration of the sample solutions
Group 1 2 3 4 5
Absorbance 2.149 0.990 0.282 0.288 0.751
Table 3: Absorbance of the unknown solution

Figure 5: Graph of Absorbance against Concentration of phosphate

6. CALCULATIONS
6.1 Using Beer’s Law equation
From the graph the equation of the line of best fit was found to be y=0.3726x. Note that the line was made to pass through the origin as when the concentration of phosphate was 0 mg/L, the spectrophotometer showed zero absorbance. This was due to the blank sample which was used to zero the spectrophotometer.
The concentration of phosphate in each of the five unknown solution was found using Beer’s Law equation: A= ?cl where in the form of y=mx+c , the gradient of the line is defined by ?l which was equal to 0.3726.
This equation can be rewritten in the form c=A/0.3726 where c is the concentration of phosphate in the unknown solution and A is the absorbance.
The results are shown in the table below:
Unknown sample solution A B C D E
Absorbance 2.149 0.990 0.282 0.288 0.751
Concentration of phosphate (mg/L) 5.77 2.66 0.757 0.773 2.02
Table 4: Concentration of phosphate in the unknown solution
6.2 Error analysis
Errors were introduced throughout the whole experiment making the results less accurate. This was shown by the graph where the value of R2 was not close to the expected value of 1. Human errors could be that the vials were not properly cleaned with tissue paper before placing them in the spectrophotometer. When the PhosVer? 3 powder pillow reagent was added to the vial, not all of its content was mixed with the solution. Instrumental errors such as in the spectrophotometer where the prism or the slits (see Figure 1) could have been displaced a little leading to inaccurate absorbance values. Errors in the pipettes used to measure 10 ml of each solution (sample, blank and unknown) were made.
Percentage error of each pipetting using the 10 ml pipette = 0.05/10 ×100%
= 0.5%
7. DISCUSSION OF RESULTS
7.1 Colour formation
It was observed that keeping the amount of molybdate and ascorbic acid (both found in the PhosVer? 3 powder pillow) constant, the intensity of the blue colour was observed to be proportional to the concentration of phosphate in the solution (Douglas R., 2007) as shown in the figure below:
Light blue
Least amount of phosphate Dark blue
Greatest amount of phosphate
Figure 6: Results on completion of the chemical reaction
(http://public.iorodeo.com/docs/phosphate/hach_phosver3.html)
Note: 1 ppm = 1 mg/L
The blue solution obtained indicated the presence of phosphate and the blue colour was due to the formation of the element molybdenum (Mo).
7.2 Plotting the graph
The calibration curve was drawn with a straight line so that Beer’s Law equation (A= ?lc) could be applied. This formula was valid only if the absorbance did not exceed the value of 1. Over that value the relationship between concentration and absorbance deviate considerably from Beer’s Law.

7.3 Values obtained in the experiment
From Figure 5, the value of R2 obtained (R2 = 0.7442) deviated largely from the expected value of 1. This was due to interfering substances which lead to inaccurate absorbance values.
The absorbance obtained for unknown solution A (Absorbance = 2.149) was greater than 1. The relationship between absorbance and transmittance became non-linear (Stockford I.M., 2007) meaning that Beer’s Law was no longer applicable to determine the concentration of phosphate in unknown solution A.
The concentration of phosphate (mg/L) in unknown solution A, B and E was 5.77, 2.66 and 2.02 respectively. If these amounts were found in water courses which normally should be about 0.02 mg/L, it would result in eutrophication in the long term.

8. CONCLUSION
The spectrophotometric blue method was very effective to find the concentration of phosphate in an unknown solution. The intensity of the blue colour showed visually which of the solutions contains the least phosphate content and which contains the greatest phosphate content. Amount of orthophosphate in the unknown solution was determined using a calibration curve of absorbance against known concentration of orthophosphate and using the application of Beer’s Law (A=?lc). The experiment could be repeated on a large scale such as testing for the presence of phosphate in wastewater. This would be useful in the future to know at what rate phosphorus increases in water.

9. REFERENCES
ABBASI, S.A., 1998. Water Quality Sampling and Analysis. New Delhi: Discovery Publishing House

ADELOWO, F.E. et al, 2016. MAYFEB Journal of Environmental Science. The spectrophotometric Evaluation of Phosphate in soil sample. online, 1 (20-29)
Availablefrom:www.mayfeb.com/OJS/index.php/ENV/article/download/80/48Accessed31October 2017

BHUTIA, T.K., 2016. Quantitative chemical analysis. online
Available from: https://www.britannica.com/science/quantitative-chemical-analysis Accessed 22 October 2017

DABKOWSKI, B. AND WHITE, M., 2016. Understanding the Different Phosphorus Tests. online
Available from: https://commonwealthengineers.com/wp-ontent/uploads/2017/05/Hach-May-2016-Phosphorus-Testing-Handout.pdf Accessed 31 October 2017

DOOLITTLE, P., 2014. Ascorbic Acid Method For Phosphorus Determination. online
Available from: http://community.asdlib.org/activelearningmaterials/files/2014/06/Lake_Study_Ascorbic_Acid_Method_for_Determining_Phosphorous.pdf Accessed 28 October 2017

DOUGLAS, R., 2007. New Mexico Wastewater Laboratory Certification Study Guide. online, 11
Available from: https://www.env.nm.gov/swqb/documents/swqbdocs/UOCP/StudyManuals/WWLabStudyGuide/WWLabStudyGuide.pdf Accessed 29 October 2017

GREEN, J., 2017. How Do Phosphates Affect Water Quality?. online
Available from: https://sciencing.com/phosphates-affect-water-quality-4565075.html Accessed 31 October 2017

HEDA, N., 2013. Spectrophotometry – UV-Visible Spectrophotometry. online
Available from: http://namrataheda.blogspot.com/2013/07/spectrophotometry-part-2-uv-visible.html Accessed on 31 October 2017

HELMENSTINE, A.M., 2017. Beer’s Law Definition and Equation. online
Available from: https://www.thoughtco.com/beers-law-definition-and-equation-608172 Accessed 29 October 2017

MURPHY, S., 2007. General Information on Phosphorus. Online.
Available form: http://bcn.boulder.co.us/basin/data/NEW/info/TP.html Accessed 28 October 2017

STOCKFORD, I.M., 2007. Reduction of error in spectrophotometry of scattering media using polarization techniques. online
Available from: https://www.ncbi.nlm.nih.gov/pubmed/18198032 Accessed 31 October 2017

TAJER, A., 2016. What’s the function of Phosphorus (P) in plants?. online
Available from: https://www.greenwaybiotech.com/blogs/news/whats-the-function-of-phosphorus-p-in-plants Accessed 31 October 2017
(Chemistry For Engineers Practical Handout)