COLORIMETRIC METHOD FOR PHOSPHATE ANALYSIS IN WATER SAMPLES PDF

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COLORIMETRIC METHOD FOR PHOSPHATE ANALYSIS INWATER SAMPLES1 Learning ObjectivesUpon completing this lab practical and writing its report you should be able to: - Practice good chemistry laboratory practices: prepare solutions using volumetric and graduated glassware, use a micro-pipette, a scale and...

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COLORIMETRIC METHOD FOR PHOSPHATE ANALYSIS IN WATER SAMPLES 1 Learning Objectives Upon completing this lab practical and writing its report you should be able to: • Practice good chemistry laboratory practices:

•

s Use a c Be aware of area-specific S i.e. in this case Standard Methods for the Examination of Water and Wastewater) Understand the principles of a c and of

•

Apply p

• •

and

Lab Objective Your task in this experiment is to play the role of a water scientist or engineer and You will use the colorimetric method known as the Ascorbic Acid Method for Phosphate analysis described in the “Standard Methods for the Examination of Water and Wastewater” [1]. You will need to: • • and • ples • Discuss (quantitatively and qualitatively) h and 2 Background Phosphate is an essential element in many processes, including for growing food and for sustaining life, as it is present even in our DNA molecules.

Since 1905, the Standard Methods for the Examination of Water and Wastewater contains hundreds of the best available, generally accepted procedures for analysing water, wastewater and related materials. The technique most commonly used is This is a method based on a The principle behind this is that e ( ) and a m tartrate ( ) react in an with dilute solutions o to form an intensely c ( 𝑷𝑶 𝟑− + 𝑴𝒐𝑶 + 𝟏𝟐𝑯 𝑶 . This complex is reduced 𝟏𝟐𝑴𝒐𝑶 𝟐− to an intensely bl

(reducing agent) (Eq. 2) and the (Figure 1).

𝑷𝑶𝟒𝟑− + 𝟏𝟐𝑴𝒐𝑶𝟒𝟐− + 𝟐𝟕𝑯+ → 𝑯𝟑 𝑷𝑶𝟒 𝑯𝟑 (𝑴𝒐𝑶𝟑 )𝟏𝟐 + 𝟏𝟐𝑯𝟐 𝑶

Eq. (1)

𝑯𝟑𝑷𝑴𝒐𝟏𝟐𝑶𝟒𝟎 + 𝒓𝒆𝒅𝒖𝒄𝒊𝒏𝒈 𝒂𝒈𝒆𝒏𝒕 → [𝑯𝟒 𝑷𝑴𝒐𝟖𝑴𝒐𝟒 𝑶𝟒𝟎 ]𝟑−

Eq. (2)

Figure 1: Standards with different concentrations of phosphate yielding different shades of blue. Courtesy of IORodeo [1]

Spectrophotometric methods rely on passing a beam of light through your sample and (Figure 2).

Figure 2: Illustration of the principle of spectrophotometric measurements

According to Beer-Lambert law (Eq. 3), the ). ,ε -1

,𝑙

and

𝐴 = 𝑙𝑜𝑔10

𝐼0 𝐼

=𝜀×𝑙 ×𝐶

(Eq. 3)

In this experiment, .

3 Apparatus For this experiment, you will be using a spectrophotometer at 𝜆 = 880 nm, a scale, the fumehood and a few items of standard laboratory equipment and glassware, namely: Timer, Volumetric flasks, Measuring cylinder, Micropipettes and volumetric glass pipettes, sample pots, disposable spectrophotometer cuvettes.

4 Experimental Protocol IMPORTANT There will be 2 . Therefore you should all work together as a team. You should share the responsibilities of making the solutions and measuring all the calibration standards and the samples. At the end you should also share all your results. Due to high number of operators in the experiment, make sure to note down exactly what you did and how you did it, e.g.: mass of reagents, units, order of procedures, volumes of solutions, any observations in terms of colouring or other and finally the operator’s name.

1. Calibration Curve Preparation In total there will be , for construction of the calibration curve. • • Each student will prepare 2 standard solutions (100 mL) by . The concentrations will be as follows: o Student A: 0.1, 1.5 ppm – P o Student B: 0.4, 1.0 ppm- P o Student C: 0.7, 2.0 ppm- P. • Don’t forget to label each standard with concentration, date, name of operator (who prepared it).

2. Reagent Solutions Preparation Each group will prepare their own reagent solution as follows: • Sulfuric Acid Solution: 5 . • Antimony potassium tartrate solution: . Dilute to a volume of 50 mL. • Ammonium molybdate solution: Dilute to a . • Ascorbic acid solution: Di . • A demonstrator will take each group in turn to a fume hood to prepare the reagent solution. To prepare 1

o o o o

50 mL 5N H2SO4, *note: the demonstrator must do this addition step* 5 mL antimony potassium tartrate solution, 15 mL ammonium molybdate solution, 30 mL ascorbic acid solution

3. Standards and sample analysis You will now individually analyse the two standards that you have prepared, the unknown water sample and a blank (using a sample of demineralised water instead of a regular sample).

To analyse each standard/sample/blank, to a sample container: • Add 800 μL of combined reagent solution • Add 5 mL of sample/standard/demineralised water (blank) - note the time/begin timing • Mix thoroughly • Add 2 mL of each sample/standard/blank to a spectrophotometer cuvette. Each group only needs to prepare one cuvette for reading per sample/standard/blank. Group 1 will provide Replicate A: Duplicate 1, and Group 2 will provide Replicate B: Duplicate 1 for a total of 2 readings per sample/standard/blank in the table below. • 15 minutes after having added the reagent solution to your sample, read the absorbance values using the spectrophotometer T (after 15 min). . Discuss this with the demonstrator before starting. 4. Washing up Wash all glassware u

uid. R

5 Analysis of Results Share the results with all the members of your session by writing them on the whiteboard. In total you will have two readings for each standard. Collate all your results in a table resembling this one: Date of the experiment

Stock Solution P

All operator names and group number

Mass of KH2PO4 (g) Volume (mL) Concentration (ppm-P) Absorbance readings Replicate A

Standards ppm-P

operator name group# (specify repl. A/B)

Replicate B

and Duplicate 1

Duplicate 2

Duplicate 1

Duplicate 2

Duplicate 1

Duplicate 2

0.1 0.4 0.7 1.0 1.5 2.0 Origin and date

-

-

Blank

-

-

Sample 1

-

-

1. Using all the data collected, plot the calibration curve (along with error bars) and assess its fit to the experimental data (R2). What is the equation for your calibration curve and what is the uncertainty associated with each value? (Tip – In Excel, use the LINEST function). 2. Determine the precision of the different standards measured using the principles of error propagation and discuss potential sources of error. 3. Discuss the role of the blanks, what it means and how it should be used in the context of a calibration curve. 4. Calculate the concentrations of phosphate of the different samples and present them in a table with their associated experimental uncertainties. Discuss if the results were expected by finding resources in literature that indicate typical phosphorus concentrations in similar samples.

6 References/Supporting material

[1] APHA, AWWA, & WPCF. (1995). Standard methods for the examination of water and wastewater. In A. D. Eaton, L. S. Clesceri, & A. E. Greenberg (Eds.), Standard Methods for the Examination of Water and Wastewater. Baltimore: Port City Press. [2] IORodeo, Smart Technology, Pasadena US, https://iorodeo.com/, [last accessed 20-092017] [3] Harris, D. (2007). Quantitative chemical analysis (7th ed.). New York: W. H. Freeman. -> for further material on error analysis and Beer-Lambert law. Linest function: [4[ Harvey, D, 2020. Using Excel for a Linear Regression [Online]. LibreTexts. Available from: https://chem.libretexts.org/Courses/Providence_College/CHM_331_Advanced_Analytic al_Chemistry_1/05%3A_Standardizing_Analytical_Methods/5.06%3A_Using_Excel_for _a_Linear_RegressionURL [01/10/2020]....