AIMS AND OBJECTIVES
The aim of this experiment is to determine the concentration of phosphate in a cola sample using
spectrophotometry, utilizing the formation of molybdovanadophosphoric acid as the basis for analysis. To
achieve this aim, specific objectives have been outlined. These include diluting the cola sample to an
appropriate concentration range, preparing a series of standard phosphate solutions to construct a
calibration curve, and ensuring precise addition of reagents to facilitate the formation of the
molybdovanadophosphoric acid complex. By accomplishing these objectives, the experiment aims to
accurately quantify the phosphate content in cola samples, which is essential for assessing its nutritional
composition and quality.
INTRODUCTION
Phosphate, an inorganic derivative of phosphorus commonly added to processed foods like sodas, plays a
crucial role in stabilizing acidity and alkalinity, thereby enhancing taste and extending shelf life.
However, excessive phosphate levels have been linked to accelerated aging and vascular damage,
underscoring the importance of precise regulation in food processing. Spectrophotometry, facilitated by a
spectrophotometer, emerges as a reliable method for quantifying phosphate concentration in cola samples.
Spectrophotometry operates on the principle of measuring the absorption of light by a substance. A
spectrophotometer, an instrument widely used in analytical chemistry, measures the intensity of light
passing through a sample solution across different wavelengths. The amount of light absorbed by the
sample at specific wavelengths correlates with the concentration of the target substance—in this case,
phosphate. By analysing the absorption spectrum, spectrophotometry enables accurate determination of
phosphate levels, providing valuable insights into nutritional composition and quality control in beverage
production. This experiment aims to employ spectrophotometric techniques to assess the phosphate
content in colas, shedding light on its role in product formulation and consumer health.
EXPERIMENT PROCEDURE
A 10 mL cola sample was pipetted into a 100 mL volumetric flask. Deionized water was then added to fill
the flask up to the mark, and the solution was mixed well by inverting the volumetric flask twelve times.
Three cola samples for this experiment were prepared by pipetting 5 mL of the solution prepared above
into three different 100 mL volumetric flasks. These samples were then set aside. A 20 μg/mL standard
phosphate solution was prepared in a 250 mL volumetric flask. Using a 50 mL burette, different volumes
(0, 8.0, 16.0, 24.0, 32.0, and 40 mL) of the diluted phosphate solution were added into six different 100
mL volumetric flasks. To each of the nine volumetric flasks (the three cola samples and the six phosphate
standard solutions), 10 mL of nitric acid solution was firstly added, followed by 10 mL of vanadate
solution, and lastly, 10 mL of molybdate solution. The 10 mL solutions (nitric acid, vanadate, and
molybdate) were added using a 10 mL pipette. This formed molybdovanadophosphoric acid with a P:
V:Mo ratio of 1:1:11. Deionized water was then added into each of the nine volumetric flasks to fill them
up to the mark, and each flask was mixed well by inverting it twelve times. The solutions were left to
react for 20 minutes.
After 20 minutes, the absorbance of the six phosphate standard solutions and the three cola samples was
measured using a spectrophotometer at a wavelength of 400 nm. Firstly, the cuvette was rinsed three
times with deionized water. Then, it was filled with deionized water, and the transparent sides of the
cuvette were wrapped with a soft tissue. The cuvette was then inserted into the spectrophotometer to
measure the absorbance of the deionized water. The cuvette was handled carefully to avoid any damage
by holding it on the sides with vertical lines using the tip of the fingers. Secondly, the absorbance of the
first standard solution with 0 mL of phosphate was measured using the same procedure of rinsing the
cuvette three times with the solution and wrapping the transparent sides of cuvette with a soft tissue
before inserting it in the spectrophotometer. However, the spectrophotometer was zeroed at the first
solution so that only the absorbance of phosphate was measured. Subsequently, the absorbance of the
other phosphate standard solutions and the three cola samples was measured using the same procedure
and the results were recorded.
RESULTS
MASS OF PHOSPHATE IN STANDARD SOLUTIONS
1. 𝑚 = 𝐶 × 𝑉
𝑚 = (20𝜇𝑔 𝑚𝐿−1 ) × (0.0𝑚𝐿)
𝑚 = 0.0𝜇𝑔
2. 𝑚 = 𝐶 × 𝑉
𝑚 = (20𝜇𝑔 𝑚𝐿−1 ) × (8.0𝑚𝐿)
𝑚 = 160.0𝜇𝑔
3. 𝑚 = 𝐶 × 𝑉
𝑚 = (20𝜇𝑔 𝑚𝐿−1 ) × (0.0𝑚𝐿)
𝑚 = 320.0𝜇𝑔
4. 𝑚 = 𝐶 × 𝑉
𝑚 = (20𝜇𝑔 𝑚𝐿−1 ) × (24.0𝑚𝐿)
𝑚 = 480.0𝜇𝑔
5. 𝑚 = 𝐶 × 𝑉
𝑚 = (20𝜇𝑔 𝑚𝐿−1 ) × (32.0𝑚𝐿)
𝑚 = 640.0𝜇𝑔
6. 𝑚 = 𝐶 × 𝑉
𝑚 = (20𝜇𝑔) × (40.0𝑚𝑙)
𝑚 = 800.0𝜇𝑔
TABLE 1: Absorbance of different volumes added.
STANDARD PHOSPHATE
SOLUTION
1
2
3
4
5
6
VOLUME OF PHOSPHATE IN
SOLUTION
(mL)
0
8.0
16.0
24.0
32.0
40.0
MEASURED ABSORBANCE
AT 400nm
VOLUME OF PHOSPHATE IN
SAMPLE
(mL)
5.0
5.0
5.0
MEASURED ABSORBANCE
AT 400nm
0.000
0.046
0.097
0.138
0.193
0.240
TABLT 2: Absorbance of samples.
SAMPLES
1
2
3
0.105
0.104
0.104
GRAPH OF ABSORBANCE AT 400nm VERSUS MASS OF PHOSPHATE
0.3
ABSORBANCE AT 400nm
0.25
y = 0.0003x - 0.0011
R² = 0.9991
0.2
0.15
0.1
0.05
0
0
-0.05
100
200
300
400
500
600
MASS OF PHOSPHATE (𝜇𝑔)
700
800
900
MASS OF PHOSPHATE IN EACH REPLICATE SAMPLE
SAMPLE 1
𝑦 = 0.0003𝑥 − 0.0011
-Absorbance of the first sample is equal to 0.105, so 𝑦 = 0.105
0.105 = 0.0003𝑥 − 0.0011
𝑥=
0.105 + 0.0011
0.0003
𝑥 = 353.67𝜇𝑔
⸫ Mass of Phosphate in sample 1 is 353.67𝜇𝑔
SAMPLE 2 & 3
-Sample 2 and 3 have the same absorbance, equal to 0.104.
𝑦 = 0.0003𝑥 − 0.0011, where 𝑦 = 0.104
0.104 = 0.0003𝑥 − 0.0011
𝑥=
0.104 + 0.0011
0.0003
𝑥 = 350.33𝜇𝑔
⸫ The mass of Phosphate in sample 2 and 3 is 350.33 𝜇𝑔
MEAN OF PHOSPHATE’S MASS
𝑥̅ =
353.67𝜇𝑔 + 350.33𝜇𝑔 + 350.33𝜇𝑔
3
𝑥̅ = 351.44𝜇𝑔
ERROR IN PHOSPHATE’S MASS
(𝑥1 − 𝑥̅ )2 + (𝑥2 − 𝑥̅ )2 + (𝑥3 − 𝑥̅ )2
𝐸𝑅𝑅𝑂𝑅 = √
3
(353.67 − 351.44)2 + (350.33 − 351.44)2 + (350.33 − 351.44)2
𝐸𝑅𝑅𝑂𝑅 = √
3
𝐸𝑅𝑅𝑂𝑅 = ±1.57 𝜇𝑔
⸫The mass of phosphate is 351.44±1.57 μg
THE CONCENTRATION OF PHOSPHATE IN COLA SAMPLE
For sample:1
[𝑃𝑂4 2− ] in aliquot =
=
𝑀𝐴𝑆𝑆 𝑂𝐹 𝑃𝐻𝑂𝑆𝑃𝐻𝐴𝑇𝐸 𝐼𝑁 𝑆𝐴𝑀𝑃𝐿𝐸
𝑉𝑂𝐿𝑈𝑀𝐸 𝑂𝐹 𝑇𝐻𝐸 𝑆𝐴𝑀𝑃𝐿𝐸
353.67
100
= 3.54 ppm
Concentration of Phosphate in diluted cola sample(C1):
𝐶1 × 𝑉1 = 𝐶2 × 𝑉2
𝐶1 (5𝑚𝐿) = (3.54𝑝𝑝𝑚)(100𝑚𝐿)
𝐶1 = 70.80 𝑝𝑝𝑚
Concentration of phosphate in cola sample(c1):
𝐶1 × 𝑉1 = 𝐶2 × 𝑉2
𝐶1 (10𝑚𝐿) = (70.80𝑝𝑝𝑚)(100𝑚𝐿)
𝐶1 = 708 𝑝𝑝𝑚
For sample:1 & 2
[𝑃𝑂4 2− ] in aliquot =
=
𝑀𝐴𝑆𝑆 𝑂𝐹 𝑃𝐻𝑂𝑆𝑃𝐻𝐴𝑇𝐸 𝐼𝑁 𝑆𝐴𝑀𝑃𝐿𝐸
𝑉𝑂𝐿𝑈𝑀𝐸 𝑂𝐹 𝑇𝐻𝐸 𝑆𝐴𝑀𝑃𝐿𝐸
350.44
100
= 3.50 ppm
Concentration of Phosphate in diluted cola sample(C1):
𝐶1 × 𝑉1 = 𝐶2 × 𝑉2
𝐶1 (5𝑚𝐿) = (3.50𝑝𝑝𝑚)(100𝑚𝐿)
𝐶1 = 70 𝑝𝑝𝑚
Concentration of phosphate in cola sample(c1):
𝐶1 × 𝑉1 = 𝐶2 × 𝑉2
𝐶1 (10𝑚𝐿) = (70𝑝𝑝𝑚)(100𝑚𝐿)
𝐶1 = 700 𝑝𝑝𝑚
Concentration of phosphate in cola in all 3 sample:
Sample1: 708 ppm
Sample2: 700 ppm
Sample3: 700 ppm
Standard deviation = 3.77
Mean = 702.67
%RSD= 0.54%
⸫ The concentration of phosphate in cola is 702.67 ± 3.77 ppm
CALCULATING THE %PHOSPHATE IN COLA:
1 𝜇𝑔 = 10−6 𝑔
%𝑃ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 =
𝑚
× 100
𝑣
SAMPLE:1
%𝑃ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 =
353.67 × 10−6
× 100
10 × 10−3
= 3.54%
SAMPLE:2 & 3
%𝑃ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 =
350.33 × 10−6
× 100
10 × 10−3
= 3.50%
Calculating %Relative error
17 𝑚𝑔 = 17000 𝜇𝑔
17000 𝜇𝑔
94.97𝑔.𝑚𝑜𝑙−1 (𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒)
True value of phosphate in the cola sample = 100×10−3 𝑙 × 30.97𝑔.𝑚𝑜𝑙−1 (𝑝ℎ𝑜𝑠𝑝ℎ𝑜𝑟𝑢𝑠)
= 521 ppm
%𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑒𝑟𝑟𝑜𝑟 =
702.67𝑝𝑝𝑚 − 521𝑝𝑝𝑚
× 100
521𝑝𝑝𝑚
= 34.87%
DISCUSSION
The concentration of phosphate in the three cola samples was determined to range from 700 ppm to 708
ppm, with a mean of 702.67 ppm and a relative standard deviation (%RSD) of 0.53%. This low %RSD
indicates high precision and consistency among the results, suggesting adherence to proper experimental
procedures such as cuvette handling, standard solution preparation, and accurate spectrophotometry
utilization. The calibration curve exhibited nearly linear behaviour with a correlation coefficient of
0.9991, indicating successful preparation and measurement of standard solutions. This further validates
the reliability of the experimental process. However, a relative error of 34.87% was observed in the
concentration of phosphate in cola samples. This discrepancy suggests potential errors during the
experiment, possibly occurring during the preparation of the cola samples.
CONCLUSION
Based on the findings, although the experiment showcased precision and adherence to protocol, the
notable relative error suggests that there's a need for enhancement in sample preparation techniques.
Therefore, future experiments could greatly benefit from a more meticulous approach to sample
preparation, aiming to minimize errors and improve result accuracy. By leveraging spectrophotometric
methods, researchers can continue to advance our understanding of the nutritional composition and
quality control measures in beverage production, contributing to improved consumer health and product
development.
References
Bellefonds, C. D. (2019, frebuary 21). What to Know About Phosphates, the Food Additive That’s in
(Almost) Everything You Eat. Retrieved from well+good:
https://www.wellandgood.com/what-are-phosphates-in-food/
Vo, K. (2023, march 9). Spectrophotometry. Retrieved from LibreTexts chemistry:
https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_M
aps/Supplemental_Modules_(Physical_and_Theoretical_Chemistry)/Kinetics/02%3A_React
ion_Rates/2.01%3A_Experimental_Determination_of_Kinetics/2.1.05%3A_Spectrophotome
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Whelan, C. (2018, september 18). Sodium phosphate. Retrieved from healthline media:
https://www.healthline.com/health/sodium-phosphate