Faculty of Engineering and

the Built
Environment
Department of
Chemical
Engineering
Advanced
Diploma
(ADCHME)

Applied chemistry 4: Analytical and environmental

ACH470S

Semester 1- 2022

UV SPECTROSCOPY
Determination of phosphate in water samples using UV Spectroscopy
Experiment 3
Group H

Initials and Surname Student number

BE Moreetsi 219265291
L Mondi 215149947
A Mtywaru 218032684
ST Ncanana 219039658
Date of report submission 18 May 2022
Grade

Except for the support provided by the teaching staff, I confirm that this is my own work. I
declare that I will not give this assignment to another student.

Signature (student):… .....Date: 18/05/2022

Due date: Wednesday, 18 May 2022 @ 23:59

Abstract
In this experiment, the concentration of KH2PO4 in water samples is determined using UV
Spectroscopy method. The absorbance of the samples is measured, and a calibration curve
is plotted to help determine the concentration of KH2PO4 in a river water sample. The results
obtained show that the concentration of KH2PO4 in the samples decreases with an increase in
concentration. This is not an expected result based on the Beer-Lamberts law.

It was then concluded that Beer's Law does not hold for the determination of KH2PO4 in river
water sample.

i
Contents
Abstract ................................................................................................................................................... i
1. Introduction.................................................................................................................................... 1
1.1. Phosphorus ........................................................................................................................... 1
1.2. Sources of phosphorus ....................................................................................................... 1
1.3. Methods used to determine phosphate in water ............................................................. 2
1.3.1. Molybdenum spectrophotometric method ................................................................ 2
1.3.2. UV-visible method ........................................................................................................ 2
2. Literature review ........................................................................................................................... 3
2.1. Aims and objectives ................................................................................................................. 4
3. Procedure ...................................................................................................................................... 5
3.1. Setting up instruments ......................................................................................................... 5
3.2. List of reagents supplied ..................................................................................................... 5
3.3. Sample preparation .............................................................................................................. 5
3.4. Preparation of CALIBRATION STOCK STANDARD ...................................................... 5
3.5. Preparation of solutions for curve calibration method .................................................... 5
3.6. Analysis of samples and standards ................................................................................... 5
4. Results ........................................................................................................................................... 6
5. Discussion and Conclusion......................................................................................................... 8
6. Reference list ................................................................................................................................ 9
Annexure A.......................................................................................................................................... 10

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1. Introduction
1.1. Phosphorus
Phosphorus is an important nutrient that is found in abundance in nature. It is one of the
most important elements for plant and animal growth. It is the earth's eleventh most
prevalent element, and it is most usually found as phosphate. In watery bodies, phosphorus
is required for microbial growth. The main cause of eutrophication in surface water is a rise
in phosphate concentration (Pradhan and Pokhrel, 2013).

1.2. Sources of phosphorus

Both natural and man-made sources of phosphorus can leak into the environment. Rocks,
minerals, and sediment are natural sources, while fertilizers and detergents are man-made
sources. Many jurisdictions in the United States have prohibited phosphate detergents,
forcing most detergent manufacturers to reformulate their products. Many fields utilize
phosphate-based fertilizers because they are very effective at producing crops (Macháček
and Malát, 1981).

Any rain that falls on a field will run off into a body of water, resulting in high phosphorus
levels. This can be particularly problematic in agricultural areas like the central valley, where
a huge section of the land is fertilized, resulting in large rises in phosphate levels in nearby
rivers and streams. The figure below shows how phosphate naturally enters water sources,
such as rivers and lakes (Macháček and Malát, 1981).

Figure 1 Schematic showing the phosphorus cycle (Macháček and Malát, 1981)

Heavy algal development occurs when phosphate is present in water, which is undesirable.
As a result, phosphorus determination is essential for water analysts and limnologists. Large

1
amounts of phosphorus have been utilized in detergents, fertilizers, and the sugar industry
(Pradhan and Pokhrel, 2013).

1.3. Methods used to determine phosphate in water

• Molybdenum spectrophotometric method
• UV-visible method
1.3.1. Molybdenum spectrophotometric method
A well-known technique for detecting phosphate is the molybdenum blue spectrophotometric
method. It includes forming molybdophosphoric acid from ortho phosphate and an excess of
molybdate in an acidic solution, then reducing it to get molybdenum blue. The absorbance of
the molybdenum blue produced in this way is measured spectrophotometrically at a
wavelength that yields the highest absorbance. The intensity of the blue colour is related to
the amount of phosphate in the sample solution (Pradhan and Pokhrel, 2013).

1.3.2. UV-visible method

UV/visible (UV-Vis) spectroscopy is an analytical technique that measures the concentration
of analytes of interest by measuring the molecular absorption of light radiation. Visible and
ultraviolet light radiation (energy) is absorbed or released when electronic transitions
between energy levels in compounds occur. In the spectra for the compounds, the strengths
of energy absorption or emission associated with transitions between the relevant electronic
energy levels are shown (Clark, 2020).

2
2. Literature review
The transitions are between an empty nonbonding or anti-bonding orbital and a bonding or
lone pair orbital. In light radiation, the relationship between frequency (v) and wavelength ()
can be thought of as waves made up of perpendicular, oscillating electric and magnetic
fields (Clark, 2020).

𝑣λ = c c is the speed of light in a vacuum (3 x 108 m/s)

Equation 1 Relationship between wavelength and frequency (Clark, 2020)

𝐸 = ℎ𝑣 h = Planck's constant (6.626 x 10-34 J)

Equation 2 Energy (Clark, 2020)

The strength with which electrons are pulled to the nuclei of the connected atoms
determines the amount of energy necessary to make an electronic transition in a molecule.
As a result, the characteristic energy of a transition is a property of a group of atoms or
chromophores (the system of electrons that absorb UV — visible light). Once metal ions
have been coupled with an adequate ligand to generate a visible complex, UV/Visible
spectroscopy can be used to measure their concentration in solution (Clark, 2020).

Basic principles for measuring the UV-visible spectrum: The terms transmittance and
absorbance are used to describe beam attenuation. When a light beam passes through a
solution, its electromagnetic radiation strength is reduced owing to absorption (Clark, 2020).
The transmittance T of a solution is defined as the fraction of incident radiation it transmits,
as shown by the equation:

𝐼
𝑇=
𝐼0

Equation 3 Transmittance (Clark, 2020)

The following equation represents the absorbance of a solution:

𝐼0
𝐴 = 𝑙𝑜𝑔10 𝑇 = 𝑙𝑜𝑔
𝐼

Equation 4 Absorbance of a solution (Clark, 2020)

Bouguer and Lambert showed that the fraction of incident light absorbed is independent of
the intensity of the source, i.e., absorption A is dependent on the "path-length" b of the
measuring cuvette. Beer demonstrated that absorption is proportional to the number of
molecules absorbing (or concentration c)

𝐴 𝛼 𝑏 and 𝐴 𝛼 𝑐

3
The combination gives rise to Beer — Lamberts Law:

𝐴 = 𝑎𝑏𝑐

Equation 5 Beer-Lamberts’s equation (Clark, 2020)

The units employed for b and c will visibly influence the quantity and dimensions of a, where
an is the proportionality constant (absorptivity).

The cell pathway length in cm is b

The concentration is expressed in mol/L or g/L.

When the concentration is provided in moles per litre and the cell length is in cm, the
absorptivity is called molar absorptivity and bears the symbol. In this experiment, the amount
of phosphate in a water sample is quantified using a curve calibration approach.

2.1. Aims and objectives

• To have a better understanding of the UV-VIS Spectrophotometric Analysis Method's
principle and operation.
• Understand how to apply the Beer-Lambert law to quantitative analysis.
• Determine the phosphate concentration in a river water sample using a UV-Visible
spectrometer.

4
3. Procedure
3.1. Setting up instruments
The UV/VIS spectrophotometer was set at an appropriate wavelength using the instructions
provided in the OPERATING MANUAL. The lamb was given time to warm up.

3.2. List of reagents supplied

1. Potassium dihydrogen phosphate (dried at 100°C)
2. Vanadate molybdate colour reagent (Prepared by dissolving 3.60 grams of sodium
vanadate in 800 ml of cold water, then adding 48.0 grams of sodium molybdate
dehydrate, and diluting to the mark in a 1L volumetric flask).
3. River Water Samples
3.3. Sample preparation
50 cm of a sample was degassed by heating it gently in a measuring cylinder and set aside
to cool. 10 cm3 of the degassed sample was pipetted into a 200 cm3 volumetric flask and
diluted to the mark by distilled water.

3.4. Preparation of CALIBRATION STOCK STANDARD

In a volumetric flask containing 1 cm3 perchloric acid, about 0.1 grams of KH2PO4 were
weighed to the nearest 0.0001 gram and dissolved in 500 mL of water. The concentration of
P in a stock solution was calculated in ppm.

3.5. Preparation of solutions for curve calibration method

Pipette 25 cm3 aliquots of each prepared standard into separate 50 cm3 volumetric flasks
(five standard solutions and one blank). As a blank, a flask containing deionized water was
employed. Each of the six volumetric flasks received 10 cm3 vanadate molybdate colour
reagent and 3 cm3 perchloric acid. Each flask was diluted to the mark using deionised water
and mixed well, then waited 10 minutes for colour development to occur. CAUTION: When
pipetting the colour reagent, gloves should be used because it includes NH4VO3, a probable
mutant.

3.6. Analysis of samples and standards

Parameters were set as per Teaching Assistant’s instruction, in the “’scan” mode under
”setup’’. The instrument was zeroed with the blank in both cuvettes, and then a spectrum of
between 300-700 nm was obtained for the most concentrated standards. The wavelength
corresponding to the maximum absorbance. The wavelength was set to “maximum” in the
“concentration” mode, and all the other parameters were setup according to the given
instructions. Each solution's absorbance was measured at a certain wavelength. The
absorbance of the blank samples was tested ten times and recorded. NOTE: Adjust the
dilutions if the sample readings fall outside the calibration range.

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 4. Results
• The table below shows the calculated concentration of KH2PO4 at different volume,
calculations are shown in annexure A.

Volume (mL) Concentration (M) Absorbance

1 0.73 0.029
2 0.35 0.034
5 0.14 0.073
10 0.07 0.156
Table 1 Calculated concentration of KH2PO4

• The calibration curve of the samples is plotted below.

Absorbance vs Concentration
0.18
0.16
0.14
0.12 y = -0.1543x + 0.1237
Absorbance

0.1
0.08
0.06
0.04
0.02
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Concentration (M)

Figure 2 Calibration curve of standards

• The table below (table 2) shows the measured absorbance of 5 samples and 1 blank
sample (table 3).

Absorbance
Trial Sample 1 Sample 2 Sample 3 Sample 4 River water
1 0.029 0.034 0.071 0.159 0.088
2 0.028 0.034 0.073 0.159 0.088
3 0.029 0.035 0.075 0.154 0.088
4 0.029 0.035 0.074 0.154 0.088
5 0.029 0.034 0.074 0.153 0.087
Average 0.029 0.034 0.073 0.156 0.088
Table 2 Sample absorbance

• The table below shows the absorbance of a blank sample recorded 10 times.

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 Blank sample Absorbance
1. 0.054
2. 0.057
3. 0.056
4. 0.056
5. 0.055
6. 0.057
7. 0.055
8. 0.057
9. 0.058
10. 0.056
Table 3 Blank sample absorbance

• The table below shows the calculated mean, standard deviation and relative standard
deviation of the samples.

Samples Mean Standard deviation RSD

0.329 0.295 0.897

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 5. Discussion and Conclusion
One factor that determines a sample's absorbance is its concentration (c). According to the
report's literature, as the concentration rises, more radiation should be absorbed, and the
absorbance should rise. As a result, according to Beer-Lamberts' equation, concentration is
proportional to absorbance. The findings of this experiment contradict the preceding
premise; the concentration of samples calculated in table 1 shows that the concentration of
the samples increases as the concentration decreases.

As a result, Beers law does not apply to the assessment of KH2PO4 in river water samples.

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6. Reference list
1. Clark, J. (2020). The Beer-Lambert Law. [online] Chemistry LibreTexts. Available at:
[Link]
ook_Maps/Supplemental_Modules_(Physical_and_Theoretical_Chemistry)/Spectros
copy/Electronic_Spectroscopy/Electronic_Spectroscopy_Basics/The_Beer-
Lambert_Law.
2. Macháček, V. and Malát, M. (1981). A new extraction method for the
spectrophotometric determination of phosphates. Microchemical Journal, 26(3),
pp.307–315. doi:10.1016/0026-265x(81)90105-3.
3. Pradhan, S. and Pokhrel, M.R. (2013). Spectrophotometric Determination of
Phosphate in Sugarcane Juice, Fertilizer, Detergent and Water Samples by
Molybdenum Blue Method. Scientific World, [online] 11(11), pp.58–62.
doi:10.3126/sw.v11i11.9139.

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Annexure A
Mr (KH2PO4) = 136.086 g/mol

Calculating moles of KH2PO4

𝑚
𝑛=
𝑀𝑟

0.1 𝑔
𝑛=
136.086 𝑔/𝑚𝑜𝑙

𝑛 = 0.0007 𝑚𝑜𝑙

Calculating concentration of KH2PO4

𝑉 = 1 𝑚𝐿 (Given in the manual)

𝑛
𝐶=
𝑉

0.0007
𝐶=
0.001

𝐶 = 0.73 𝑀

• The same method was used to calculate the concentration at 𝑉 = 2 𝑚𝐿, 𝑉 = 5 𝑚𝐿 𝑎𝑛𝑑

𝑉 = 10 𝑚𝐿 and the results are tabulated in table 1.

• The concentration obtained and absorbance of samples are plotted on the figure
below and used to calculate the concentration in the river sample.

Absorbance vs Concentration
0.18
0.16
0.14
0.12 y = -0.1543x + 0.1237
Absorbance

0.1
0.08
0.06
0.04
0.02
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Concentration (M)

𝑦 = −0.1543𝑥 + 0.1237

• Using beer-lambert equation

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 𝐴 = 𝑎𝑏𝑐

𝐴 = 0.088 (Average absorbance of river sample)

𝐴 = −0.1543𝑥 + 0.1237

0.088 = −0.1543𝑥 + 0.1237

𝑥 = 0.23 𝑀

Mr (Phosphate) = 94.97 g/mol

0.23 𝑚𝑜𝑙 94.97 𝑔 1 𝑚𝑔

𝑥= × × −3 = 2.2 × 104 𝑝𝑝𝑚
𝐿 𝑚𝑜𝑙 10 𝑔

• There are 2.2 × 104 𝑝𝑝𝑚 of phosphate ions in the river sample

LOD and LOQ based on 3𝜎 rule

From the calibration curve, we take the slope of the blank solution as the skewness =
0.48.

∑ 𝑥𝑖
𝑥̅ =
𝑛

0.73 + 0.365 + 0.146 + 0.073

𝑥̅ =
4

𝑥̅ = 0.329

∑(𝑥𝑖 − 𝑥̅ )2
𝜎= √
𝑛−1

(0.73 − 0.329)2 + (0.365 − 0.329)2 + (0.146 − 0.329)2 + (0.073 − 0.329)2

𝜎= √
4−1

𝜎 = 0.295

𝜎
𝑅𝑆𝐷 =
𝑥̅

0.295
𝑅𝑆𝐷 =
0.329

𝑅𝑆𝐷 = 0.897

3𝜎
𝐿𝑂𝐷 =
𝑠𝑙𝑜𝑝𝑒

3(0.295)
𝐿𝑂𝐷 =
0.48

𝐿𝑂𝐷 = 1.844

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 10𝜎
𝐿𝑂𝑄 =
𝑠𝑙𝑜𝑝𝑒

10(0.295)
𝐿𝑂𝑄 =
0.48

𝐿𝑂𝑄 = 6.146

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