Overview

In this experiment you will determine the phosphorus (P) concentration of an aqueous sample using a standard method of analysis from Standard Methods for the Examination of Water and Wastewater.

To cite this lab manual: “Ascorbic Acid Method for Phosphorus Determination”. A Manual of Experiments for Analytical Chemistry. Department of Chemistry at UW- Madison, Summer 2024.

Visual Abstract

Background

Since 1905, Standard Methods for the Examination of Water and Wastewater¹ has been the standard text for water analysis. It contains hundreds of the best available, generally accepted procedures for analyzing water, wastewater, and related materials. The Standard Methods document describes three possible chemistries for measuring P: the vanadomolybdophosphoric method, the stannous chloride method, and the ascorbic acid method. The technique most commonly used is the ascorbic acid method. In this method, ammonium molybdate (NH₄)₆Mo₇O₂₄·4H₂O) and antimony potassium tartrate (K(SbO)C₄H₄O₆·½H₂O) react in an acid medium with dilute solutions of orthophosphate to form an intensely colored antimony-phospho-molybdate complex. This complex reduces to an intensely blue-colored complex by ascorbic acid² and the absorbance of the complex is measured at 880 nm. In this experiment, you will use the ascorbic acid method to determine the concentration of phosphorus in a water sample.

Lab Concept Video

Write down observations or notes from the video in your lab notebook.

Pre-lab Work

Lab Skills

Review these lab skills videos prior to lab.

Extra Resources:

• Using the LabQuest to Collect Spectrophotometry Data
• Making Scientific Plots
• Finding Property Data

Prelaboratory Exercises

In your lab notebook, include a detailed purpose and procedure (including the list of equipment you will need), as well as responses and calculations to solve the following problems.

1. Your group will need to prepare a stock solution of phosphorus (P) from which you will prepare standard phosphorus solutions. How many grams of KH₂PO₄ (MW = 136.09) must be weighed out to make 100 mL of a 50 mg P/L stock solution? What is this concentration in mmol P/L?
2. After reading the experimental procedure below, create a table in your laboratory notebook that describes how you will use the stock P solution and subsequent dilutions to prepare a set of standard solutions of P ranging in concentration from 0.1 mg/L to 2.0 mg/L. Plan to prepare a minimum of 6 standards.

Before You Take The Quiz on Canvas

• Understand how to determine the concentration of an unknown solution using Beer’s Law.
• Determine the concentration of standard phosphorus solutions needed to prepare a calibration curve suitable for determining your unknown concentration.
• Know what a blank is and how to use it in spectrophotometric measurements.
• Calculate the concentration of phosphorus in a solution prepared from potassium dihydrogen phosphate (KH₂PO₄).
• Understand the relationship between percent transmittance and absorbance.
• Understand how a spectrophotometer works.

Experimental

The procedure in the text boxes below is copied directly from the Standard Methods for the Examination of Water and Wastewater. Working with your lab partners, you will be adapting the methods described to develop a working procedure in our lab space for measuring total phosphorus in a lake water sample. Some of the modifications and choices you’ll need to make have to do with the available equipment we have in the lab. Other choices have to do with the limitations of the experiment, which are described in the text below. The standard method text is shared on the left column of the page; guidance and considerations in your decision-making are provided on the right column of the page. You will also decide an appropriate range of concentrations for the standards. There exists no singular “right” answer to the procedure. The “right” procedure will result in a sound and reliable method for measuring phosphorus in a lake water sample.

In the end, have a clear experimental plan drafted in your lab notebook before the beginning of lab. In talking with your lab partners, you might have some differences in the details. Many times, those differences are simply a result of different choices in initial concentrations or dilution ratios; the results will work out fine. Sometimes the differences are because of a math error, or perhaps misinterpretation of the text. It’s important to talk out any differences in advance of entering the lab to minimize problems with the procedure. Be specific in how you will make solutions, including how much KH2PO4 you plan to weigh out and what the final concentrations of your solutions will be after dilution. Write your procedure in your notebook and follow it carefully while you perform your analysis.

Standard Method Text

Manual Guidance

Note in section 1-c the concentration ranges recommended based on the path length of the cuvet. The SpectroVis plus and Ocean Optics spectrometers we have in the lab use 1 cm cuvets. Consider your answer in question 2 of the prelaboratory exercise when planning the standard concentrations. Section 2-b points to section on acid washed glassware. The referenced section is shared below. We will provide 1 M HCl in the lab. Warm this on the hotplates and rinse only the necessary glassware with small aliquots to prepare for this lab.(Section referenced in 2-b) 4500-P.C.2b. Acid-washed glassware: Use acid-washed glassware for determining low concentrations of phosphorus. Phosphate contamination is common because of its adsorption on glass surfaces. Avoid using commercial detergents containing phosphate. Clean all glassware with hot dilute HCl and rinse well with distilled water. Preferably reserve the glassware only for phosphate determination, and after use, wash and keep filled with water until needed. If this is done, acid treatment is required only occasionally. You will need to prepare Reagent a (5 N H2SO4) using 9 M H2SO4, instead of concentration H2SO4. Normality, N, is moles of reactive unit per liter. For H2SO4, H+ is the reactive unit, so 1 M H2SO4 is 2 N H2SO4. Reagents b, c, and d will be prepared for you. When preparing Reagent e, the “Combined Reagent”, the precursors must be added in the order listed and mixed after each reagent is added for the proper chemistry to occur. Make sure you have recorded this information correctly in your notebook. Reagent f. The directions ask you to prepare 1 L of this stock solution; you only need to prepare 500 mL of this solution. DON’T FORGET TO MAKE REAGENT G (standard phosphate solution in step g). While the standard method asks you to record absorbance, you should record percent transmittance (%T) at 880 nm and convert %T values to absorbance using the relationship A = -log T (or A = 2-log(%T)). It is important to measure the percent transmittance for all samples after the same time interval (i.e., consistently measure %T 15 minutes after addition of the combined reagent). Instructions on how to use the LabQuest spectrophotometer and save results are on Canvas.

Create a table like the one below in your laboratory notebook. Fill in the rows titled “Expected Concentration”, “Standard P Solution (mL)”, and “Distilled H₂O”, with the values you plan to use to make your standards. The sum of the volumes of Standard Phosphate Solution you use plus the amount of distilled H₂O should add up to 50 mL. Since this table simply reflects a plan, note that you will derive the “Actual Concentration” when you record the exact mass of KH₂PO₄ weighed out on the balance in lab.

You may find your group will make several shifts in the procedure as you begin preparing solutions for the experiment. In fact, this practice is a very normal event when developing a method for standard analysis. Take good notes on WHY a particular change or shift happened. For example, “The solution turned blue upon adding the combined reagent to the blank sample” might mean there was phosphorous contamination, either in the RO water you used or residual on your glassware. Your macroscopic observations are important data points in learning what does or doesn’t work. Be patient with the process! Taking good notes about your thoughts now will serve your group well later on, as you begin further modifications to the experiment for other purposes.

Post-Lab Work Up

Results/Calculations

Fill out the answer sheet for this experiment completely. Answer the following post-lab questions.

1. Calculate the absorbance from the measured percent transmittance values and determine an average absorbance value for each of the solutions measured.
2. Use EXCEL to prepare a calibration curve of average absorbance versus phosphorus concentration. Make sure to include the point from the blank you prepared. Turn in a copy of your calibration curve along with your notebook pages and answer sheet.
3. Using the calibration curve, determine the concentration of phosphorus in your unknown solution. Report the concentration of the unknown in units of mg P/L and mmol P/L.

Challenge Questions

Challenge questions are designed to make you think deeper about the concepts you learned in this lab. There may be multiple answers to these questions! Any honest effort at answering the questions will be rewarded.

1. Lake water samples can sometimes contain particulate matter, sludge, algae, and other complexed chemicals that result in colored or cloudy (turbid) samples. Describe two possible modifications to the methods above to accommodate samples that are colored or turbid.

Lab Report Submission Details

Submit your lab report on Canvas as 1 combined PDF file.  This submission should include:

1. The completed answer sheet.
2. Your lab notebook pages associated with this lab, which should include answers to the post-lab questions and challenge questions.

The grading rubric can be found on Canvas.

References:

1. Eaton, A.D.; Clesceri L.S.; Rice E.W.; Greenberg A.E.; Franson M.H., Eds., Standard Methods for the Examination of Water and Wastewater: 21^st ed.; American Public Health Association: Washington, DC, Water Environment Federation: Alexandria, VA, and American Water Works Association: Denver, CO, 2005.
2. Murphy, J.; Riley, J.R. A modified single solution method for the determination of phosphate in natural waters. Anal. Chem. 1977, 27, 31-36.
3. Harris, D. C. & Lucy, C. A. Quantitative Chemical Analysis, 10^th ed.; W. H. Freeman: New York, NY, 2020