EXPERIMENT 9:

DETERMINATION OF PHOSPHATES

A Laboratory Report Presented to the

Faculty of the Department of Chemical Engineering
School of Engineering and Architecture
Saint Louis University

In Partial Fulfillment of the Requirements for the Course:

CHE 3131L: Environmental Engineering for CHE (Laboratory)

by:
Cudia, Genery Mae P.
De Guzman, Marriane Camille J.
Del Rosario, Jehra Luz J.
Dial, Katherine Joyce A.

November 2020
 Department of Chemical Engineering
School of Engineering and Architecture
Saint Louis University

LABORATORY REPORT EVALUATION SHEET

Laboratory Course: CHE 3131L_____ Schedule: 1:30-6:00 WTh_____
Experiment Number: __9__
Experiment Title: DETERMINATION OF PHOSPHATES
Group Number: __7__ Date Performed: December 14, 2020
Group Members: Cudia, Genery Mae P. Date Submitted: December 21, 2020
De Guzman, Marriane Camille J.
Del Rosario, Jehra Luz J.______
Dial, Katherine Joyce A._______

CONTENTS TOTAL REMARKS SCORE

POINTS
I. Abstract 15

II. Introduction 10

III. Design and Methodology 10

IV. Results and Discussions 15

V. Conclusion and 10
Recommendation

VI. Appendices: 15
a. List and Uses of
Apparatus
b. Definition of Terms
c. Documentation
d. Computations
e. Answers to
Questions/Problems

VII. Table of Contents/ List of 10

Tables/ List of Figures
VIII. Bibliography/References 5
(APA format)

IX. Format and Neatness 10

TOTAL POINTS: 100 SCORE: ________

Evaluated by: Engr. Gizelle Pascua Date: ___________
 ABSTRACT

Phosphate is an electrically charged particle which contains the mineral

phosphorous that is responsible for algal bloom at high concentration. Determination of
phosphate is important for checking water quality. This experiment used the ascorbic acid
method, and the product of the reaction in this method undergoes spectrophotometric at
880 nm. The purpose of this experiment was to determine the amount of phosphate in
the sample water, based on the formation of heteropoly acid—phosphomolybdic acid due
to the reaction between ammonium molybdate and antimony potassium tartrate with
orthophosphate followed by its reduction with intensely colored molybdenum blue in
ascorbic acid medium. The result showed the following sulfate concentrations: 0.0891,
0.0993, and 0.0742 for trials 1, 2, and 3 respectively. With trial 3 as the lowest, and trial
2 as the highest. Therefore, the result agrees with the statement of the linear relationship
between the calculated absorbance and concentration. The calculated average was of
0.0875 mg P/L. Therefore, the students concluded that the water sample it is not safe for
drinking.
KEYWORDS: Phosphate, Phosphorus, Ascorbic Acid Method, Spectrophotometric
i
TABLE OF CONTENTS
CHAPTER I: INTRODUCTION ...................................................................................... 5
CHAPTER II: DESIGN AND METHODOLOGY ............................................................. 7
CHAPTER III: RESULTS AND DISCUSSION ............................................................... 8
CHAPTER IV: CONCLUSION AND RECOMMENDATION ......................................... 12
APPENDICES ............................................................................................................. 13
REFERENCES ............................................................................................................ 17

LIST OF TABLES
TABLE 9.1: DATA OBTAINED FOR THE STANDARD CURVE.....................................8
TABLE 9.2: DATA OBTAINED FOR THE DETERMINATION OF PHOSPHATES.........8
TABLE 9.3: DATA FOR CALIBRATION CURVE………………………………………….10
TABLE 9.3: DATA AND RESULTS……………………………………………………........11

LIST OF FIGURES
Figure 9.1: STANDARD CALIBRATION CURVE………………………………………….9
Figure 9.2: CALIBRATION CURVE………………………………………………………...10
 INTRODUCTION

Phosphorus is the eleventh most abundant element on earth surface commonly

found as phosphates. It is vital nutrient to living organism, and it stimulates growth of the
aquatic plant and algae. But, this becomes a major pollutants in water. Phosphates
originates from sources such as human and animal waste, detergents and food residues.
Municipal wastewaters may contain from 5 to 20 mg/l of total phosphorous, of
which 1-5 mg/l is organic and the rest in inorganic. The individual contribution tend to
increase, because phosphorous is one of the main constituent of synthetic detergents.
The individual phosphorous contribution varies between 0.65 and 4.80 g/inhabitant per
day with an average of about 2.18 g(Lenntech,n.d.). The Agricultural is one major
contributor of phosphate in a natural or man-made way of contributing.
High levels of phosphates in aquatic environments can fuel algal growth, resulting
in algal blooms that can potentially lead to eutrophication as the thick algal mats block
out sunlight causing the algal cells to die off. Oxygen is stripped from the water column
as the dead algae cells decompose, leading to anoxic conditions that can result in mass
die-offs of fish and other aquatic life (Miley, 2018) Phosphate reflects BOD (Biological
Oxygen Demand), therefore the number microbes as Escherichia coli (bacterium) also
increase tremendously. The number Escherichia coli per unit volume of water is main
parameter of water pollution (Kharat, S.J & Pagar, S., 2009). Monitoring the concentration
of phosphate is essential for higher concentration may lead to environmental problems.
There are many ways in determining the phosphate but the efficiency depends on
the method they are going to use. Only few has a water treatment that could possibly
determine the presence of phosphate. The removal of Phosphate during the sewage
treatment process has become a crucial area of interest as more and more works have
Phosphorus discharge consent. With the ever increasing use of detergents containing
phosphate the problem is growing (Fosten A., 2010)
Determining the various types of phosphorus has been done throughout many
years in laboratories. Initially some was done manually but in recent time analysis has
become automated. The most common and standard method of analysis is the segment
flow analyzer and in line analyzer. The available process are based on the calorimetric
analysis of the reaction products which result in colored complex. The absorbance of
which at a specific wavelength is proportional to the concentration (based on the Beer-
Lambert Law).
The molybdenum blue phosphorous method in conjunction with UV-Visible
spectrophotometer is a very sensitive method4,5 for the determination of phosphorus at
830 nm. Phosphate in the sample can be determined at sub-ppb concentration at 830
nm. Orthophosphate and molybdate ions condense in an acidic solution to form
phosphomolybdic acid, upon selective reduction (e.g. with hydrazinium sulphate) a blue
color is produced. The intensity of the blue color is proportional to the amount of
phosphate initially incorporated into the heteropoly acid. If the acidity at the time of

5
reduction is 0.5 M in sulphuric acid and hydrazinium sulphate is the reluctant then the
resulting blue complex exhibits a maximum6 absorbance at 820-830 nm. Intensity of the
color solutions is normally measured on a visible spectrophotometer (Kharat, S.J & Pagar,
S., 2009). The method is also applicable for the determination of phosphate in nuclear
reprocessing plants, medical science, clinical science, agriculture, metallurgy and
environmental science (S Ganesh & et.al, 2012).

6
 DESIGN AND METHODOLOGY
Procedure
For the Preparation of Standard Curve:
The PO3-4 calibration standards of blank, 0.15, 0.2, 0.5, 1.0, and 1.3 mg PO 3-4 P/L
are prepared by diluting the standard phosphate solution to 50 ml. In this solution, 0.05
mL or one drop of phenolphthalein indicator was added. In line with this step, if a red color
develops, 5N H2SO4 solution should be added dropwise to discharge the color. Then, 8.0
ml of combined reagents were added, and the solution was mixed thoroughly for 12
minutes. Finally, spectrophotometric measurement was conducted.
For the Water Sample:
A 50 ml water sample was placed in an Erlenmeyer flask, then 0.05 ml of
phenolphthalein indicator was added to it. Upon the addition of the indicator, it is essential
to take note of the color that developed. If a red color develops, 5N H2SO4 solution should
be added dropwise to discharge the color. Then, 8 ml of combined reagents were added,
and the solution was mixed thoroughly for 12 minutes. Finally, spectrophotometric
measurement was conducted.
Correction for Sample Color and Turbidity of the Water Sample (high colored):
A 100 ml of water sample was placed in an Erlenmeyer flask. Then, 0.05 ml or one drop
of phenolphthalein indicator was added. Upon the addition of the indicator, it is essential
to take note of the color that developed. If a red color develops, 5N H2SO4 solution should
be added dropwise to discharge the color. Now, all reagents were added together except
for ascorbic acid and antimony potassium tartrate. Next, 8 ml of combined reagents were
added and mixed thoroughly for 12 minutes. After mixing, the spectrophotometric
measurement was conducted. In this process, the blank absorbance was subtracted from
the absorbance of each sample.
Spectrophotometric Measurement:
The wavelength was set at 880 nm. At this wavelength, the absorbance against
redistilled water with combined reagent was read and set at zero absorbance or 100%
transmittance. A sufficient amount of water sample or PO3-4 standards that were
previously treated with reagents was put in the cuvette. The cuvette was then placed in
the cuvette holder of the spectrophotometer. Finally, the absorbance was read and
recorded.

7
 RESULTS AND DISCUSSION
For Calibration Curve:

Table 9.1 Data Obtained for the Standard Curve

Concentration, mg P/L Volume of stock Absorbance reading @
phosphate solution used, 880 nm
mL
0 0 0

0.15 3 0.164

0.20 4 0.225

0.50 10 0.59

1.0 20 1.134

1.3 26 1.365

For Water Sample:

Table 9.2 Data Obtained for the Determination of Phosphates
Volume of Absorbance Concentration,
sample used, mg AVERAGE
ml reading for Concentration,
SAMPLE TRIAL P/L
mg
sample @ 880
P/L
nm
1 50 0.868 0.7937

7 2 50 0.900 0.8235 0.7924

3 50 0.832 0.7601

8
 Fig. 9.1 Standard Calibration Curve
1.6
1.365
1.4
1.134
1.2

1
Absorbance

0.8
0.59
0.6

0.4
0.225
0.164
0.2
0
0
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Concentration, mg/L

The purpose of this experiment was to determine the amount of phosphate in the
sample water, based on the formation of heteropoly acid—phosphomolybdic acid due to
the reaction between ammonium molybdate and antimony potassium tartrate with
orthophosphate followed by its reduction with intensely colored molybdenum blue in
ascorbic acid medium. The blue color produced in this method is developed by the
reduction of 12-molybdophosphoric heteropoly acid. Here the acidified solution of
phosphate is treated with ammonium molybdate that gives hetero poly acid which is then
reduced by hydrazine hydrate to give phosphomolybdenum blue i.e., a blue color
complex. The reaction involved in the formation of molybdenum blue can be given as:
PO43- + 12(NH4)2 MoO4 + 24H+ → (NH4)3 PO4 .12MoO3 + 21NH4+ + 12H2O (NH4)3 PO4
.12MoO3 + (N2H4.H2O) → molybdenum blue. The absorbance of thus formed
phosphomolybdenum blue was measured at 880 nm spectrophotometrically.
For each test for the standard curve, samples with six different concentrations, that
is, 0, 0.15, 0.20, 0.50, 1, and 1.3 mg P/L were prepared. Once the samples were
prepared, the light absorbance of each sample was monitored using the
spectrophotometer. Calibration curve is necessary to establish a relation between the
absorbance and the phosphate content for spectrophotometric analysis. The calibration
curve for the phosphate analysis is shown in Figure 7.1. The curve is obtained by plotting
absorbance as a function of concentration of phosphate (in mg P/L) at the wavelength
880 nm. The plot is linear and obeys Lambert-Beer’s law in the range of 0.15 to 1.3 mg
P/L.
For the test for water sample, total of three trials were determined
spectrophotometrically at 880 nm by molybdenum blue such as, 0.868, 0.900, and 0.832,
respectively. In each case, 50 mL of sample solution was taken followed by the addition
of the reagents under optimized conditions. Each sample was then mixed at room

9
temperature for about 12 minutes for maximum color development and hence the
absorbance was measured spectrophotometrically. Using the standard curve from the
phosphate calibration at different ammonium concentrations, values of 0.7937, 0.8235,
and 0.7601 mg P/L are plotted, with a calculated average of 0.7924 mg P/L, as provided
in Figure 7.2.
The absorbance due to PO43- against PO43- concentration of standard was plotted
against the standard calibration curve, and the sample concentrations are projected by
comparing sample absorbance with the standard curve, using the linear equation, y=
1.073x + 0.0164.

Table 9.3 Data for Calibration Curve

Concentration, mg SO4-2 / Volume of stock Absorbance Reading @

L Phosphate solution 880 nm
used, mL
0 0 0
0.15 3 0.164
0.20 4 0.225
0.50 10 0.59
1.0 20 1.134
1.3 26 1.365

CALIBRATION CURVE
1.6
Absorbance Reading @ 880 nm

1.4 y = 1.073x + 0.0164

R² = 0.9962
1.2
1
0.8
0.6
0.4
0.2
0
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Concentration, mg P / L

Figure 9.2 Calibration Curve

10
 Table 9.4 shows the results obtained in the experiment. It shows the volume of
sample used, individual absorbance reading of the sample, and the corresponding
concentration expressed in mg P / L and the average mg P / L for all trials.
Table 9.4 Data and Results

Volume
of AVERAGE
Absorbance Reading for Concentration,
Sample Trial Sample Concentration,
sample @ 880 nm mg P / L
Used, mg P / L
mL

1 50 0.112 0.0891
1 2 50 0.123 0.0993 0.0875
3 50 0.096 0.0742

The result showed the following sulfate concentrations: 0.0891, 0.0993, and
0.0742 for trials 1, 2, and 3 respectively. With trial 3 as the lowest, and trial 2 as the
highest. Therefore, the result agrees with the statement of the linear relationship between
the calculated absorbance and concentration. The calculated average was of 0.0875 mg
P / L, therefore it is not safe for drinking. This is because the reservoirs for obtaining
drinking water should not exceed 0.025 ppm or mg/L. And also, the natural
background levels of total phosphorus are generally less than 0.03 mg / L. The
natural levels of phosphate usually range from 0.005 to 0.05 mg / L. Any excess amount
of phosphate can lead to fish kills and the degradation of habitat with loss of species.
Large mats of algae can form and in severe cases can completely cover small lakes. As
a result, water can become putrid from decaying organic matter. When the concentration
of phosphates rises above 100 mg/L the coagulation processes in drinking water
treatment plants may be adversely affected. Manmade sources of phosphate include
human sewage, agricultural run-off from crops, sewage from animal feedlots, pulp and
paper industry, vegetable and fruit processing, chemical and fertilizer manufacturing, and
detergents.

11
 CONCLUSION AND RECOMMENDATION
The phosphate determination by molybdenum blue method in conjugation with UV-
visible spectrophotometer is a simple method that can be carried out in common
laboratories. The method is simple and cheap compare to other methods, because it
neither involves extraction of the analyte, nor requires any sophisticated instruments like
HPLC. The strict control of pH is not necessary in this method. In addition, the developed
new method is working satisfactorily for the determination of phosphate present in various
samples analyzed. The system obeys Lambert-Beer’s law in the phosphate concentration
range of 0.15 to 1.3 mg P/L. The work presented in this experiment reports the
optimization of reaction conditions and the concentration of the various reagents for
spectrophotometric determination of phosphate in various samples by molybdenum blue
method.
In conducting the experiment, the students recommend that the future students
should follow the procedure in a proper and orderly manner to avoid unnecessary error
and accidents. Furthermore, for improved and more accurate results, it is recommended
to make sure that the materials to be used are properly cleaned before using them to
avoid contamination, follow the procedures given in a proper and orderly manner to avoid
error and any unnecessary breakages, and ensure to conduct and finish the experiment
within the same conditions (temperature and pressure) since it will affect the results.

12
 APPENDICES

A. List and Uses of Apparatus

Apparatus Illustration Use/s

Erlenmeyer flask In this experiment, an Erlenmeyer flask was
used to mix chemical substances such as the
water sample with the necessary reagent like
the phenolphthalein indicator.

Graduated It was used to measure a specific volume of

Cylinder the reagents used in this experiment.

Rubber This was used alongside a pipette. It was used

Bulb/Pipetol as a vacuum source for filling the pipette with
the necessary reagents.

Spectrophotometer This instrument was used to determine the

(UV-Vis) concentration of phosphates in the water
sample by measuring its absorbance at a
wavelength of 880 nm.

Stopwatch This was used to make sure that the water

sample combined with the reagents were
mixed thoroughly for 12 minutes.

13
 B. Definition of Terms

Absorbance. It is also referred to as optical density, and it aims to measure the

quantity of light absorbed by a solution at a specific wavelength.
Ascorbic acid method. This is a type of colorimetric method which is based on
the reduction of ammonium molybdiphosphate complex by ascorbic acid in the
presence of antimony. The reduced compound has an intensely colored
molybdenum blue.
Orthophosphates. It is a form of phosphate produced by natural processes and
by major man-influenced sources such as partially treated and untreated sewage,
runoff from agriculture sites and application of some lawn fertilizers.

C. Documentations

(There was no documentation for this experiment because it was not done in an
actual laboratory set-up)

D. Computations
For the calibration curve:

Concentration, mg P/L Volume of stock Phosphate Absorbance reading @880

solution used mL nm
0 0 0
0.15 3 0.164
0.20 4 0.225
0.50 10 0.59
1.0 20 1.134
1.3 26 1.365

14
 STANDARD CALIBRATION CURVE
1.6
Absorbance reading at 880

y = 1.073x + 0.0164
1.4
R² = 0.9962
1.2

1
nm

0.8

0.6

0.4

0.2

0
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Concentration mg P/ L

Using the linear equation generated from the calibration of standard curve, the
absorbance of each trial and the average absorbance in mg P/L were computed.
Linear equation: y= 1.073x + 0.0164
In this equation, y is the absorbance reading for water sample at 880 nm, while x is the
concentration in mg P/L.
TRIAL 1:
Absorbance reading = 0.868
𝒚 = 𝟏. 𝟎𝟕𝟑𝒙 + 𝟎. 𝟎𝟏𝟔𝟒
𝟎. 𝟖𝟔𝟖 = 𝟏. 𝟎𝟕𝟑𝒙 + 𝟎. 𝟎𝟏𝟔𝟒
𝑷
𝒙 = 𝟎. 𝟕𝟗𝟑𝟕 𝒎𝒈
𝑳
TRIAL 2:
Absorbance reading = 0.900
𝒚 = 𝟏. 𝟎𝟕𝟑𝒙 + 𝟎. 𝟎𝟏𝟔𝟒
𝟎. 𝟗 = 𝟏. 𝟎𝟕𝟑𝒙 + 𝟎. 𝟎𝟏𝟔𝟒
𝑷
𝒙 = 𝟎. 𝟖𝟐𝟑𝟓 𝒎𝒈
𝑳
TRIAL 3:
Absorbance reading = 0.832

15
 𝒚 = 𝟏. 𝟎𝟕𝟑𝒙 + 𝟎. 𝟎𝟏𝟔𝟒
𝟎. 𝟖𝟑𝟐 = 𝟏. 𝟎𝟕𝟑𝒙 + 𝟎. 𝟎𝟏𝟔𝟒
𝑷
𝒙 = 𝟎. 𝟕𝟔𝟎𝟏 𝒎𝒈
𝑳

𝟎.𝟕𝟗𝟑𝟕+𝟎.𝟖𝟐𝟑𝟓+𝟎.𝟕𝟔𝟎𝟏
𝑨𝒗𝒆𝒓𝒂𝒈𝒆 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 = 𝟑

𝑨𝒗𝒆𝒓𝒂𝒈𝒆 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 = 𝟎. 𝟕𝟗𝟐𝟒 𝒎𝒈 𝑷/𝑳

E. Answers to Questions/Problems

1. Explain how does phosphates contributes to soil erosion in river banks?

Soil erosion is a significant contributor of phosphates in streams. When
bank scour occurs, direct removal of bank materials by the physical action of
flowing water happens. The phosphates present in the river bank are transported
in the streams or any adjacent water bodies. Hence, the decreasing amount of
phosphate in river banks increases the amount of soil erosion in such banks.

2. How do phosphates affect water quality?

Phosphorus in the form of phosphates present in excess in water bodies may
affect the water quality by causing excessive growth of algae. In this case, once
the growth of algae is out of control, water ecosystems create imbalances,
destroying other aquatic life forms, and producing harmful toxins. Moreover, the
excessive amount of algae reduces the sunlight available for the aquatic plants.
This phenomenon is called the algal bloom or the rapid increase in algae
population.

3. What is the normal phosphate level in water?

In the freshwater system, the normal levels of phosphates range from 0.005
to 0.05 mg/L. At concentration levels of 0.08 to 0.10 ppm, phosphate may trigger
periodic blooms in bodies of water. However, to ensure that long-term
eutrophication will be prevented, the total phosphorus level must be below 0.5 ppm
and 0.05 ppm, respectively.

16
 REFERENCES
Green, J. (2018, August 6). How Do Phosphates Affect Water Quality? Retrieved from
https://sciencing.com/phosphates-affect-water-quality-4565075.html
Kotoski, J. E. (1997). Retrieved from
http://osse.ssec.wisc.edu/curriculum/earth/Minifact2_Phosphorus.pdf
Oram, B. (n.d.). Phosphate in Surface Water Streams Lakes. Retrieved from
https://water-research.net/index.php/phosphate-in-water
Miley, A. (2018, July 20). Phosphorus reduction in wastewater treatment.
SustainabilityMatters.
https://www.sustainabilitymatters.net.au/content/wastewater/article/phosphorus-
reduction-in-wastewater-treatment-
1288454257#:%7E:text=When%20a%20metal%20is%20added,to%20form%20t
he%20metal%20phosphate.
Khatar, S., & Pagar, S. (2020, May 20). Determination of Phosphate in Water Samples of
Nashik District (Maharashtra State, India) Rivers by UV-Visible Spectroscopy. E-
Journal of Chemistry.
https://webcache.googleusercontent.com/search?q=cache:sZXcJSf0IJUJ:https://
downloads.hindawi.com/journals/jchem/2009/913609.pdf+&cd=2&hl=en&ct=clnk
&gl=uk
Ganesh S, Khan F, Ahmed MK, Velavendan P, Pandey NK, Kamachi Mudali U.
Spectrophotometric determination of trace amounts of phosphate in water and soil.
Water Sci Technol. 2012;66(12):2653-8. doi: 10.2166/wst.2012.468. PMID:
23109582.
Lenntech. (n.d.). Phosphorous removal from wastewater. Retrieved 18 December 2020,
from https://www.lenntech.com/phosphorous-removal.htm
Fosten, A. (2010, April 23). Analysing and Removing Phosphate in Sewage Treatment.
Envirotech Online.
https://webcache.googleusercontent.com/search?q=cache:JglK8e4Vq_AJ:https://
www.envirotech-online.com/article/water-
wastewater/9/angus_fosten/analysing_and_removing_phosphate_in_sewage_tre
atment/657+&cd=1&hl=en&ct=clnk&gl=uk

17
 DIVISION OF WORK
MEMBER WORK ASSIGNED
Cudia, Genery Mae P. Abstract, Compilation
De Guzman, Marriane Camille J. Design and Methodology, Appendices
Del Rosario, Jehra Luz. Introduction, References
Dial, Katherine Joyce A. Conclusion and Recommendations

18