QUANTITATIVE ANALYSIS OF PHOSPHATE Name

QUANTITATIVE ANALYSIS OF PHOSPHATE
Name: B. MADHOO
Student ID: 1711338
Date: 03 November 2017
University of Mauritius
REACTIVE PHOSPHORUS (ORTHOPHOSPHATE)
Determination of Phosphate
Using Spectrophotometry
From: MADHOO Bhavish
To: Mr. TOOLSY Bhoomitra
Date: 03 November 2017
ABSTRACTThe 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.

TABLE OF CONTENTS
TOC o “1-3” h z u ABSTRACT PAGEREF _Toc497433753 h 2LIST OF TABLES PAGEREF _Toc497433754 h 4LIST OF FIGURES PAGEREF _Toc497433755 h 4LIST OF EQUATIONS PAGEREF _Toc497433756 h 41. INTRODUCTION PAGEREF _Toc497433757 h 52. AIMS AND ODJECTIVES PAGEREF _Toc497433758 h 63. LITERATURE REVIEW PAGEREF _Toc497433759 h 73.1 Importance of phosphorus PAGEREF _Toc497433760 h 73.2 Types of phosphates PAGEREF _Toc497433761 h 73.3 Sources and threat of phosphates in water PAGEREF _Toc497433762 h 73.5 How to determine concentration of phosphate in water PAGEREF _Toc497433763 h 83.6 What is spectrophotometry? PAGEREF _Toc497433764 h 83.7 Application of Beer’s Law PAGEREF _Toc497433765 h 94. METHODOLOGY PAGEREF _Toc497433766 h 104.1 Apparatus used PAGEREF _Toc497433767 h 104.2 Chemicals used PAGEREF _Toc497433768 h 114.3 Procedure PAGEREF _Toc497433769 h 114.4 Determination of phosphate in the unknown solution PAGEREF _Toc497433770 h 124.5 Dangers and safety measures PAGEREF _Toc497433771 h 126. CALCULATIONS PAGEREF _Toc497433772 h 146.1 Using Beer’s Law equation PAGEREF _Toc497433773 h 146.2 Error analysis PAGEREF _Toc497433774 h 147. DISCUSSION OF RESULTS PAGEREF _Toc497433775 h 157.1 Colour formation PAGEREF _Toc497433776 h 157.2 Plotting the graph PAGEREF _Toc497433777 h 157.3 Values obtained in the experiment PAGEREF _Toc497433778 h 168. CONCLUSION PAGEREF _Toc497433779 h 169. REFERENCES PAGEREF _Toc497433780 h 1710. APPENDICES PAGEREF _Toc497433781 h 18

LIST OF TABLES TOC h z c “Table” Table 1: List of reagents, dangers and safety measures PAGEREF _Toc497431080 h 12Table 2: Absorbance of different concentration of the sample solutions PAGEREF _Toc497431081 h 13Table 3: Absorbance of the unknown solution PAGEREF _Toc497431082 h 13Table 4: Concentration of phosphate in the unknown solution PAGEREF _Toc497431083 h 14
LIST OF FIGURES TOC h z c “Figure” Figure 1: Diagram of the main components in a spectrophotometer PAGEREF _Toc497431090 h 8Figure 2: Diagram of a beam of light as it passes through the solution vial PAGEREF _Toc497431091 h 9Figure 3: Hach DR 2500 spectrophotometer PAGEREF _Toc497431092 h 10Figure 4: PhosVer 3 phosphate reagent powder pillow PAGEREF _Toc497431093 h 11Figure 5: Graph of Absorbance against Concentration of phosphate PAGEREF _Toc497431094 h 13Figure 6: Results on completion of the chemical reaction PAGEREF _Toc497431095 h 15
LIST OF EQUATIONS TOC h z c “Equation” Equation 1 : First step reaction to determine presence of orthophosphate PAGEREF _Toc497431100 h 5Equation 2 : Second step reaction to determine presence of orthophosphate PAGEREF _Toc497431101 h 5Equation 3: Beer’s Law Equation PAGEREF _Toc497431102 h 6Equation 4: Transmittance PAGEREF _Toc497431103 h 9Equation 5: Absorbance PAGEREF _Toc497431104 h 9
1. INTRODUCTIONQuantitative 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)C4H4O6.12 H2O) + Potassium
Pyrosulfate
(K2O7S2) + Orthophosphate
(PO43-)

Antimony-phospho-molybdate complex
(Yellow Colour)
Equation SEQ Equation * ARABIC 1 : First step reaction to determine presence of orthophosphateAntimony-phospho-molybdate
(Yellow Colour) + Ascorbic acid
(C6H8O6)
(Vitamin C) Molybdenum complex
(Mo)
(Blue Colour)
Equation SEQ Equation * ARABIC 2 : Second step reaction to determine presence of orthophosphateIn 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= clEquation SEQ Equation * ARABIC 3: Beer’s Law EquationA: 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 ODJECTIVESTo 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 REVIEW3.1 Importance of phosphorusPhosphorus 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 phosphatesInorganic 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 waterIn 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 waterThere 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).

center580009000
Figure SEQ Figure * ARABIC 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=ItIoEquation SEQ Equation * ARABIC 4: Transmittance-1905104775000 A= -logT= -logItIoEquation SEQ Equation * ARABIC 5: AbsorbanceIo: 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 SEQ Figure * ARABIC 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 LawThe 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. METHODOLOGYIn 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 usedHach 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 SEQ Figure * ARABIC 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 212 minute reaction (shaking for 30 seconds and 2 minutes for the reaction)
4.2 Chemicals usedPhosphate standard solution, 50 mg/L
Distilled water
1890395558165000PhosVer 3 phosphate reagent powder pillow 10 ml
Figure SEQ Figure * ARABIC 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 ProcedureProgram 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 solution10 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 measuresReagent 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 SEQ Table * ARABIC 1: List of reagents, dangers and safety measures5. 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 SEQ Table * ARABIC 2: Absorbance of different concentration of the sample solutionsGroup 1 2 3 4 5
Absorbance 2.149 0.990 0.282 0.288 0.751
Table SEQ Table * ARABIC 3: Absorbance of the unknown solution7620369760500
Figure SEQ Figure * ARABIC 5: Graph of Absorbance against Concentration of phosphate6. CALCULATIONS6.1 Using Beer’s Law equationFrom 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=A0.3726 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 SEQ Table * ARABIC 4: Concentration of phosphate in the unknown solution6.2 Error analysisErrors 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 = 0.0510 ×100% = 0.5%
7. DISCUSSION OF RESULTS7.1 Colour formation121920210629500It 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
36195328041000Figure SEQ Figure * ARABIC 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 graphThe 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 experimentFrom 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. CONCLUSIONThe 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. REFERENCESABBASI, 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)
10. APPENDICESEXPERIMENT: PHOSPHORUS, REACTIVE (ORTHOPHOSPHATE)
INTRODUCTION
Orthophosphates are highly soluble in water. The orthophosphate ion (PO43-) has the P atom centrally located and bonded to four oxygen atoms which are located at the corners of a tetrahedron. Phosphates are the primary limiting factor in fresh water plant and algal growth. Large amounts of phosphates in water stimulate excessively growth of algae leading to eutrophication. Reactive phosphorus is largely in the orthophosphate form and can be detected by a spectrophotometry method.

The PhosVer 3 (Ascorbic Acid) Method 8048 is used to detect and analyse the presence of phosphate in tap water, wastewater and seawater. This method implies that orthophosphate reacts in an acid medium with molybdate ions forming a mixed phosphate/molybdate complex. This complex is then reduced by ascorbic acid to form a highly coloured molybdenum blue. The colour intensity is found to be proportional to the amount of phosphate present in the sample solution. The absorption can be measured using a spectrophotometer to determine the amount of orthophosphate (PO43-) present.

In this experiment we will be using Beer’s Law equation to determine concentration of phosphate in an unknown solution. Beer’s Law states that the amount of photons absorbed is directly proportional to the concentration of the substance. Beer’s Law is as follows A = lc where:
A is a measure of absorbance
is the molar absorptivity
l is the optical path length
c is the concentration of the substance
This method is also called the total orthophosphate test which is largely a measure for orthophosphate and measures both dissolved and suspended orthophosphate in any unknown solution.

The range of concentration of phosphate to be used is 0.02 to 2.50 mg/L PO43- and by using a spectrophotometer a wavelength of 880 nm is selected which is suitable for the concentration of phosphate to be used in this experiment.

AIMS AND OBJECTIVES
To understand the principle of spectrophotometry and the application of Beer’s Law
To determine the concentration of phosphate in a known solution and unknown solution using a spectrophotometer
Apparatus required
Hach DR 2500 spectrophotometer
It is an instrument used to measure absorbance of light of a solution in this case a solution containing phosphate ions (PO43-)
2647951105535
50ml Beaker
It was used to store the phosphate standard solution before the solution can be pipetted.

3384552921000
10ml Pipette
To transfer 10 ml of the diluted solution into a 10 ml vial (sample solution).To transfer 10 ml of the diluted solution into a 10 ml vial (blank solution).

To transfer 10 ml of unknown solution into a 10 ml vial.

15ml Pipette
To transfer 15 ml of the phosphate standard solution into a 100 ml volumetric flask.1655445651510000568325650494000
Volumetric flask 100 ml
3943351123950It was used for accurate dilutions of the solution containing phosphate.

Vials 10 ml
They were used to store the sample solution, the blank solution and the unknown solution and placed into the cell holder of the spectrophotometer to measure absorbance.

2965453257550
Timer
It was used to set a 2 minute reaction
2927355113020
Chemicals required
Phosphate standard solution, 50 mg/L as PO4
Distilled water
PhosVer 3 phosphate reagent powder pillow 10-ml
121920793623000
Dangers while handling the following chemicals
Phosphate standard solution – In case of eye and skin contact, it can cause irritation
PhosVer 3 phosphate reagent – It contains potassium pyrosulfate and sodium molybdate which is very hazardous in case of eye and skin contact. It can cause irritation and if accidentally inhaled, it may cause respiratory tract irritation
Safety measures while handling chemicals
Wear gloves and eye protection when transferring phosphate standard solution and when adding the reagent powder pillow contents into the vial.

PROCEDURE FOR ORTHOPHOSPHATE (USING POWDER PILLOW)
Program 490 P React. PP is started using Hach DR 2500 spectrophotometer and a wavelength of 880 nm is selected.

Using a 15 ml pipette, 15 ml of the phosphate standard solution is transferred into a 100 ml volumetric flask
The volumetric flask is filled up to its mark with distilled water.

The volumetric flask is then inverted to mix the contents, thereby obtaining homogenous diluted solution.

Using a 10 ml pipette, 10 ml of the diluted solution is transferred into a 10 ml vial (sample solution)
A PhosVer 3 phosphate reagent powder pillow is cut using scissors and all its contents is added into the 10 ml vial sample solution.

The vial is immediately closed and shake vigorously for 30 seconds
After half a minute, a timer is set for a 2-minute reaction.

Meanwhile 10 ml of the diluted solution is transferred into another 10 ml vial using the 10 ml pipette (blank sample)
The vial containing the blank sample is cleaned using tissue paper and inserted into the cell holder of the spectrophotometer
The command Zero is selected to zero the spectrophotometer
After 2 minute, Step 10 is repeated for the sample solution, the command Read is selected and the absorbance is noted
The procedure is repeated for different concentration of the phosphate solution and their absorbance are noted
Determining the concentration of phosphate in the unknown solution
The solution is pipetted using a 10 ml pipette into a 10 ml vial.

The blank sample is placed in the cell holder to zero the spectrophotometer
The unknown sample is then placed in the cell holder and the absorbance level is read and noted.

TABLE OF RESULTS
Table 1: Determining the absorbance for different sample solutions
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: Determining the absorbance for the unknown solutions
Group 1 2 3 4 5
Absorbance 2.149 0.990 0.282 0.288 0.751
centerbottom00
CALCULATIONS
Equation of line obtained: y = 0.3726xBeer’s Law equation: A = cl
Gradient of line from the equation is defined by l which is equal to 0.3726
Absorbance of the unknown solution (Group 3) = 0.282, hence y = 0.282
x = c (concentration of phosphate in the unknown solution)
(0.282) = 0.3726x
x = 0.282 ÷ 0.3726
= 0.75684
?0.757 mg/L
Therefore the unknown solution has a phosphate concentration of 0.757 mg/L
From the graph the concentration of phosphate in other unknown solutions can be calculated using the equation:
Concentration of phosphate in unknown solution = Absorbance0.3726Unknown sample of each group 1 2 3 4 5
Concentration mg/L 5.77 2.66 0.757 0.773 2.02
ERROR ANALYSIS
Percentage uncertainty in 10ml pipette:
In this experiment the 10ml pipette was used twice:
To transfer 10ml of the diluted solution into a 10 ml vial (sample solution)
To transfer 10ml of the diluted solution into a 10 ml vial (blank solution)
However since the pipette was not used for both successively, the percentage error for each (i and ii) is as follows:
Percentage error for each (i and ii) using 10 ml pipette = 0.05 10 100%
=0.5%
Another 10ml pipette was used to transfer 10 ml of unknown solution into a 10 ml vial. Using this pipette, the percentage error will be the same (0.5%).

Total percentage error in the pipettes is 1.5%
Percentage uncertainty in R2 value for orthophosphate:
The value of R2 obtained from the graph is 0.7442
Percentage error = 1.00-0.74421.00 x 100%
= 25.58%
? 26%
DISCUSSION OF RESULTS
In this experiment the concentration of phosphate in the unknown solution was determined using the Ascorbic acid method 8048.

A series of dilutions were made from the phosphate standard solution and using the relationship M1V2 = M2V2. For group 3 the volume of the standard solution to be diluted was 15 ml which has a concentration of 1.5 mg/L.

Orthophosphate reacts with molybdate in an acid medium to produce a Phosphomolybdate complex. Ascorbic acid then reduces the complex, giving an intense molybdenum blue colour. The blue colour indicates that the solution contains phosphate. The intensity of blue colour is proportional to the amount of phosphate present.

A graph of absorbance against concentration of the phosphate solution is drawn. A line of best fit is drawn which passes through the origin. From the graph, the gradient of line is positive showing that the absorbance increases as concentration increases.
The values of absorbance obtained in the experiment were not the best results as the value of R2 obtained from the graph is not close to the expected value of 1.

The concentration of phosphate in the unknown solution was found to be 0.759 mg/L (Group 3)
There were also sources of error throughout the experiment:
Instrumental error such as percentage error in the pipettes used.

Human error such as forgetting to wipe off traces of fingerprints using tissue paper.

Using the same 10ml pipette for both the sample and unknown solution.

When adding the contents of the reagent powder pillow into the vial containing the solution, not all powder is mixed with the solution as some of it may fall outside the vial.

Improvement for experiment
A burette can be used instead of a pipette to transfer specific amount of the stock solution in the volumetric flask.

The 10 ml vials can be cleaned with 1:1 hydrochloric acid and rinsed with deionized water before transferring the sample solution in the vials. A detergent containing phosphate should not be used to clean the vials as it will contaminate the sample.

Before inserting the sample solution vial in the cell holder, it should be wiped with tissue paper to remove any traces of fingerprints.

The reagent powder pillow must be cut in such a way so that when adding the powder into the vial containing the solution, all of it is mixed with the solution.

Analyse sample immediately for best results. If prompt analysis is impossible, the samples should be preserved for up to 48 hours by filtering immediately and storing at below 6 0C. Before testing the sample is allowed to warm to room temperature.

CONCLUSION
The reason to carry out this experiment was to determine the concentration of phosphate in an unknown solution. By using solutions of known concentration of phosphate and a spectrophotometer, a graph of absorbance against concentration can be drawn. As a result knowing the absorbance of the unknown solutions, the concentration of phosphate in unknown solutions can be determined using the line of best fit and Beer’s Law.

REFERENCES
https://www.hach.com/asset-get.download.jsa?id=7639983836https://archive.epa.gov/water/archive/web/html/vms56.htmlhttps://msu.edu/course/lbs/171l/Phosphate.htmlhttps://www.thomassci.com/Laboratory-Supplies/Water-Quality-Test-Kits/_/PHOSVER3-PWD-PLWS10MLPK100-UN3316?q=Hach%20Powder%20Pillowshttp://www.balmann.co.kr/Details/Product/Analysis-DR2500(HACH).asphttp://www.directindustry.com/prod/hirschmann-laborgeraete-gmbh-co-kg/product-126003-1568163.html?utm_source=ProductDetail;utm_medium=Web;utm_content=SimilarProduct;utm_campaign=CAhttps://www.aliexpress.com/item-img/4Pcs-set-Graduated-Borosilicate-Glass-Beaker-Volumetric-Glassware-Chemistry-Experiment-Tool-For-Laboratory-5ml-10ml-25ml/32749804019.html#http://shop.gohcl.com/default.aspx?page=item+detail;itemcode=18493https://commons.wikimedia.org/wiki/File:Brand_volumetric_flask_100ml.jpghttp://www.piwine.com/volumetric-pipet-15ml.htmlhttp://www.boeco.com/artikelShow.php?ID=260