EXP. 9 EVALUATION OF METHODS FOR DETECTING AMMONIUM: ION SELECTIVE ELECTRODES AND SPECTROSCOPY
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1 University of Puget Sound Department of Chemistry Chem 230 EXP. 9 EVALUATION OF METHODS FOR DETECTING AMMONIUM: ION SELECTIVE ELECTRODES AND SPECTROSCOPY INTRODUCTION Nitrogen is an essential nutrient for all forms of life. Nitrogen can be used in its two major forms, reduced as urea/ammonia/ammonium ion or oxidized as nitrate ion. Urea hydrolyzes (reacts with water) in nature to form ammonia and ammonia forms an equilibrium in water with its conjugate acid ammonium ion according to its K b. In most natural water (ph below 8.75) ammonium ion is the predominant form of reduced nitrogen. Under good growing conditions, most ammonium is converted to nitrate by the urease enzyme in bacteria present in the soil. 1 Both forms of N can be utilized by plants but some prefer nitrate ion over ammonium ion. However, small changes in biologically-available nitrogen levels can dramatically affect the levels of microbiological, plant and animal life. According to the United States Department of Agriculture, the US consumed over 120 million tons of nitrogen bearing fertilizer in These fertilizers contain other nutrients, commonly as known as N-P-K (nitrogen-phosphorus-potassium), of varying percentages with nitrogen typically present in one of the reduced forms. Although helpful for plants, excess nitrogen nutrients in the ecosystem can be harmful. Eutrophication is a process where excess nutrients collect in lakes and waterways and promote excessive growth of algae called algal blooms. The growth and decay of algal blooms use up the dissolved oxygen in the water choking out fish and other aquatic animals and negatively impacts the ecosystem. The U.S. Environmental Protection Agency (U.S. EPA) has not established a maximum contaminant level for ammonia in drinking water. Environmental limits for ammonia in surface water in the US range from 0.25 to 32.5 mg/l or ppm. The National Academy of Science recommends, and many European nations have adopted, a drinking water standard of 0.5 mg/l or ppm. (NOTE: 1mg/L=1ppm) It is important to quantify nitrogen concentrations in water samples whether for drinking water safety, wastewater treatment discharge, or simply to understand how much is available in aquatic ecosystems. There exist at least seven acceptable tests for the determination of NH 3 /NH 4. Selecting a particular test procedure determines cost of the analysis (both in terms of instrumentation and personnel), speed, toxicity of reagents, sensitivity, detection limit and possible analyte interferences. Here you will compare two of these tests, a colorimetric test and an ion selective electrode test with respect to their respective sensitivities in detecting a range of concentrations of ammonium ion. ION SELECTIVE ELECTRODES Ion Selective Electrodes (ISEs) are solid state or membrane-based electrodes that measure a specific ion (e.g., Ca 2, NH 4, etc.) in an aqueous solution. When the membrane of the electrode is in contact with a solution containing the specific ion, a voltage, depending on the concentration of that ion in solution, develops at the membrane. The ISE we will use is a combination style; that is, the voltage develops in relation to an internal reference electrode (usually Ag/AgCl). The ISE we will be 1
2 using measures the ammonium ion directly. Samples need to be aqueous to avoid contaminating or dissolving the membrane. Membranes in ISEs are usually one of two types: solid polymer or solid state. The Ammonium ISE is a Solid Polymer type. In Solid Polymer membranes the membrane is a porous plastic disk, permeable to water. It allows the sensing cell to contact the sample solution and separates the internal filling solution from the sample. In Solid State Membranes a thin crystal disk is an ionic conductor. It separates the internal reference solution from the sample solution. The voltage that develops between the sensing and reference electrodes is proportional to the concentration of the reactive ion being measured. As the concentration of the ion reacting at the sensing electrode varies, so does the voltage measured between the two electrodes. The Nernst equation, describes the linear response of an ISE: where E is the measured voltage, E is the standard potential for the combination of the two half cells and is the concentration of the measured ion species, ammonium ion. Assuming the internal ionic strength, is constant, the Nernst equation may be rewritten to describe the electrode response to the concentration of the measured ionic species, NH 4 : Note that a plot of E (mv) vs. log is linear. By plotting E vs. log one can then determine the concentration of NH 4 in aqueous solutions. EXPERIMENTAL PROCEDURE WORK WITH A PARTNER. WEEK 1 A) PREPARATION OF STANDARDS AMMONIUM CHLORIDE STANDARD SOLUTIONS FOR CALIBRATION. Setup Logger Pro with the ph electrode. Calibrate the electrode with buffers ph4 and ph 10. Rinse and blot the ph electrode between solutions. 1) ph 6 Buffer Stock Solution Calculate and measure the grams of sodium acetate needed to make 900 ml of a 0.010M buffer into a 1000-mL beaker on a stir plate with stir bar. Using a 1-L graduated cylinder measure 900 ml of DI water and add to the beaker. A simple way to prepare this buffer is to monitor the ph of a sodium acetate solution with your ph electrode while you slowly add drops of 1M HCl. 2) NH 4 Standard Stock Solution (~100ppm N) Prepare a NH 4 standard stock solution by dissolving ~0.01 g (to nearest g) of NH 4 Cl (FW 53.49}, dried at 100 C, in your prepared ph 6 Buffer in a 250-ml volumetric flask. This should get you a standard that is ~100 ppm (2) (3) 2
3 (N). Transfer to a clean labeled container. You will use this stock solution to make the rest of your standards. 3) Standard NH 4 Dilutions Using your 100 ppm ammonium ion (NH 4 ) standard stock solution, 10-, 5-, and 1-ml pipets, and a 50-mL volumetric flask, prepare five (5) standard diluted solutions that cover a concentration range from the stock solution (100 ppm) to 1ppm. Remember to use your ph 6 buffer solution to make each dilution and NOT DI water. Transfer to clean labeled test tubes with stoppers and store in a test-tube rack with your name and your partner s names. Prepare three (3) more standard dilutions from 1ppm to 0.01 ppm (this is 8 total standards including the stock and covers 5 orders of magnitude!). Store any remaining ph 6 buffer in your cleaned and rinsed 1-L poly bottle for next week. The following procedure will be done in the second week of the experiment WEEK 2 B) PREPARATION OF SOLUTIONS FOR PHENATE METHOD Obtain the following 4 stock solutions in clean labeled test tubes: 1 ml of MnSO 4 stock solution. 5 ml of the bleach stock solution in a clean test tube (bleach is NaOCl, this solution has been acidified to make hypochlorous acid HOCl). 5 ml of phenate reagent in a clean test tube (This is a 10% solution of phenol with 2.5% NaOH). 50 ml of 100ppm stock KCl in a clean test tube. With a volumetric pipet add 10mL of your standard NH 4 dilutions to 8 labeled large test tubes (start with lowest concentration first). Be very careful about cross contamination. Prepare 2 additional test tubes, one with 10mL of just buffer (blank) and another with 10 ml of 100 ppm KCl (the interfering ion). Add one drop of MnSO 4 to each test tube. With a micropipet add ml of the bleach solution to each test tube. Switch tips, adjust the micropipet and quickly add ml of the phenate solution to each test tube. Swirl each test tube gently, place the rack of tubes out of the way and record the time in your notebook. These solutions need to react for 60 minutes. NH 4 DETECTION ION SELECTIVE ELECTRODE (ISE) METHOD USING ISE CALIBRATION CURVE Soak the Ammonium ion-selective electrode probe in the 100ppm stock solution in the plastic vial for 30 minutes. Find and start the ISE Logger Pro template on your computer to record Potential (mv) vs. [N]ppm(N) (we will measure based on ppm N since it is constant regardless of the form of ammonia/ammonium). Make a new calculated column for log [N]. Rinse a glass weighing vial and half fill with lowest concentration of your standard 0.01ppm solution. Start Collection Using a ring stand, place the ammonium ion-selective electrode probe in the vial. To ensure proper readings keep the ISE from 3
4 touching the vial and make sure that the salt bridge plug is submerged. Hold the ISE still and swirl the vial gently, then let the solution sit 60 sec and record the voltage measurement as mv. Record the E (mv) for each of your standard NH 4 solutions, moving from lowest concentration solution (~0.01ppm) to highest concentration (100ppm). Rinse and gently blot the ISE between solutions. Do not wipe the liquid membrane tip or surface of an ion selective electrode which may cause damage or contamination to the sensing element. Plot the E (mv) versus log[n] (ppm) and perform a linear regression on the plot. Save a copy of your calibration curve with the linear regression line to include in your report. If your plot tails off, include all points in your plot but attempt 2 or 3 regression lines excluding outlying points. These line equations and correlations should all be recorded and discussed in your final report. Selectivity of the ISE Once you have saved your ISE calibration plot, use the real time ISE mv digital window and CLEARLY in your notebook record the mv of the 100ppm KCl solution. Calculate the concentration of NH 4 in ppm that this solution would assigned. NH 4 DETECTION PHENATE METHOD USING SPECTROSCOPIC CALIBRATION CURVE After an hour has passed, close Logger Pro, disconnect the ISE, and reopen Logger Pro (no template) with a new file and the spectrometer connected. Calibrate the spectrometer with the blank (buffer with reagents but no ammonium). Dump the blank and fill the cuvette with the 100ppm stock solution and take an absorbance spectrum. Save the Absorbance spectrum. Set Logger Pro to record λ max vs. concentration (in ppm). Your λ max should be near 630nm. Rinse the cuvette with buffer and then rinse once with your lowest concentration (~0.01ppm) solution. Dump the rinse solution and refill with lowest ppm solution and record the absorbance at λ max for this concentration. Also record this data in your notebook in a table. Repeat the rinse and measurement steps for all of your standards and the KCl interfering ion. Save a copy of your calibration curve with the linear regression line to include in your report. If your plot tails off, include all points in your plot but attempt 2 or 3 regression lines excluding outlying points. These line equations and correlations should all be recorded and discussed in your final report. REFERENCES (1) Molins-Legua,C.; Meseguer-Lloret,S.; Moliner-Martinez,Y.;Campins-Falco,P. A Guide for Selecting the Most Appropriate Method for Ammonium Determination in Water Analysis. Trends Anal. Chem. 2006, (2) (3) Vernier Software and Technology. Ion Selective Electrodes, 6. 4
5 WHAT TO DO To Be Turned in From Exp. 8- Electrochemistry: Copy pages of completed Tables 2-4 from Part 1 with Sample Calculations. Copy pages with the table and labeled diagrams (with descriptive captions) of the two batteries you constructed in Part 2, with the electrodes labeled (anode and cathode) and the half reaction occurring at each electrode. Include the voltage measured and the calculated voltage for the half-cells you used. Copy page with the answer for Question 1. Question.1. In Part 2A, steps 2 and 3, you replaced the metal electrodes with graphite. The two reactions occurring in a cell are: M a (s) M a (aq) e - (aq) M b (aq) e - (aq) M b (s) Which reaction is occurring at the red wire. For Experiment 9 WEEK 1 Before Lab: Read this experiment and Harris, Chapter 14 Sections 1-6. Set up your lab notebook appropriately for this experiment including a title bar, a purpose and the Table of Reagents (should include the following reagents; ammonium chloride, sodium acetate trihydrate, acetic acid, hypochlorous acid, sodium hydroxide, phenol, and manganese(ii) sulfate, anhydrous). Be sure to note the disposal procedures in your table (all of our solutions can be poured down the drain with running water). You should also include the following information: CALCULATIONS (Show all calculations clearly in notebook.): (1) Preparation of acetate buffer: Calculate the amount of sodium acetate and 1M HCl needed to make 0.90 L of a M acetate buffer at ph 6 (2) Preparation of 100 ppm ammonium chloride solution: Calculate the amount of ammonium chloride (NH 4 Cl) required to make 250 ml of a solution that is 100 mg/l (or parts per million, ppm) NH 4 as N. (There are 14.0g N/mol NH 4.) (3) Prepare a dilution scheme to make the standards in Part A, Step 3. Determine which pipets you will use and calculate the concentration of standards that you will make to satisfy the standards requirement in Part A, Step 3. Show a dilution scheme and calculations in your notebook. WEEK 2 Instructions for the second week preparation will be in this spot next week. Be sure to check next week for the WEEK 2-What-to-Do WEEK 1-During the Lab: Carry out the procedure as written. Modify your procedure if you find it necessary, but be sure to record the modifications. Complete your calculations and graphs and enter relevant data in your lab notebook. 5
6 Record all other pertinent observations and measurements as you do the experiment. Show appropriate sample calculations and sketches in your laboratory notebook. To Be Turned In NEXT WEEK (from WEEK 1): A table with the concentrations of your Standard NH 4 solutions with columns for results for the two methods. The data for the ISE will have units of mv and for the Phenate method the data will be Absorbances. To Be Turned In For the Completed Experiment - AFTER SECOND WEEK This is a formal lab report including a brief introductory paragraph stating the purpose of this lab in a few sentences as well as Results, and Discussion. Treat the report as an analysis of the two ammonium detection methods. You should include all pertinent information needed in determining which method to use for what level of ammonium. You should include a discussion on the relative pros and cons of each method such as sensitivity and interferences. The Results should include calibration curves and data tables that help tell the story that you are trying to tell and provide the reader. Your figures and tables should be included within the body of the text with ample introduction. Attach your copy pages with sample calculations. 6
WEEK 2 EXP. 9 EVALUATION OF METHODS FOR DETECTING AMMONIUM: ION SELECTIVE ELECTRODES AND SPECTROSCOPY
University of Puget Sound Department of Chemistry Chem 230 WEEK 2 EXP. 9 EVALUATION OF METHODS FOR DETECTING AMMONIUM: ION SELECTIVE ELECTRODES AND SPECTROSCOPY INTRODUCTION Nitrogen is an essential nutrient
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