Page 1 of 10 v 2004 Photo of Brown’s River at Mill’s River Park, Jericho VT Photo by Jack Cameron How Much Stuff Is In There?: Quantitative Analysis of Phosphate In Natural Waters Objectives: . Learn how to perform a quantitative spectrophotometric assay; Analyze data using the statistical and graphing functions of Excel; Determine the level of free phosphate in a pond water sample. Introduction: Scientists ask a lot of questions. We ask: “What kind of stuff is there in this piece of the universe we are studying? Is that a neutrino being emitted from the sun? What compounds are present in drinking water? Are there sodium ions in this softened water? Is there water on Mars? Are there anabolic steroids in the urine of major-league baseball players? What elements are present? Is mercury present in this lake perch? Is this shiny nugget gold or iron?” This is qualitative analysis. Once you know what stuff is present, the next question you want to ask is: “How much stuff is there in this piece of the universe we are studying? How many neutrinos are being emitted from the sun? How much sodium ion is present in the softened water? How much phosphate is going into the lake from the new fish hatchery?” This is quantitative analysis, from the Latin quantus?, “How much?” In this lab you will quantitatively determine the amount of phosphate in a water sample taken from a pond bordering the SMC campus or the Winooski River. ON PHOSPHORUS IN WATER: Phosphorus, the element, is non-metallic, and exists in three forms, or allotropes: These are red, white and black phosphorus. Elemental phosphorus exhibits some rather interesting chemistry. For example, when white phosphorus is placed in the dark and is exposed to air, it glows green and gives off white smoke. In warm, humid air it will spontaneously burst into flames. Ingestion of as little as 50-100 mg of white phosphorus may cause death. That being said, phosphorus-containing oxides, phosphates, (PxOyn-) are absolutely necessary for life as we know it. As water runs over and through rocks it carries off small amounts of phosphates. These inorganic phosphates (phosphates not attached to carbon-containing compounds) are a plant nutrient and are taken in by plants with water and incorporated into organic phosphate compounds. Animals obtain phosphorus from phosphates in water and plant material. Clean streams and lakes have a phosphorus concentration of approximately 0.02 ug/L (parts per million, ppm). Phosphates are essential for algae and plant growth, and at the 0.02 ug/L level, they are the limiting reagent for plant growth. Add more Page 2 of 10 v 2004 nitrogen of potassium (also essential for plant growth) and plant and algae levels don’t change. Add more phosphate, and you end up with a lot more green stuff in the lake. Adding phosphates to lakes and stream increases eutrophication – that is, excessive growth of algae and plants, resulting in the depletion of oxygen in the water. Eutrophication can lead to fish kills, degradation of habitat, and loss of species. Large mats of algae can form and sometimes completely cover small lakes or shallow bays. Sources of excess phosphate include human sewage, agricultural run-off from crops, sewage from animal feedlots, fish hatcheries, the paper industry, vegetable and fruit processing, chemical and fertilizer manufacturing, and detergents. Phosphorus and Suspended Material Phosphorus is present in streams and lake waters in several different forms: O orthophosphate, PO43-; polyphosphates [PnO3n+1(n+2)-] and metaphosphates P Phosphate ion, [(PO3)nn- ]; and organic phosphorus. Orthophosphate is the most biochemically O- -O O- “orthophosphate” available form of phosphorus in natural waters, and microorganisms catalyze the hydrolysis of condensed phosphates to orthophosphate. However, 30 to 60 O percent of the phosphorus present in many natural waters is bound to organic O P material. O O- O O P O- P O O- P O O O- P O- -O O O- P O- O -O O- A polyphosphate H3O+ O- O O P O Hydrolysis of a O O- polyphosphate to -O P 3 P P orthophosphate O- O- -O O- O- O- O C N C NH H C O C C O N N NH 2 P P -O O- CH 2 O O O- O- C H H C C C H H O H P O- -O O- Page 3 of 10 v 2004 One may analyze phosphorus content in water several ways, and get very different results. A Total Phosphorus analysis, for example, will measure phosphate, polyphosphates, metaphosphates, and organic phosphates, both dissolved in the water and adsorbed onto suspended solids. This is a somewhat time-consuming procedure, requiring reaction of the samples with strong acids, and heating. In this experiment, we will test only for soluble orthophosphate, that is, PO43- dissolved in the water sample. See: Merck Index, 9th Edition, 1976. M. Windholtz, ed. Merck and Company, Rahway, NJ, Groundwater Contamination: Sources, Control, and Preventative Measures. 1989. Chester D. Rail, ed. Technomic Publishing Co., Inc., Lancaster, PA. http://va.water.usgs.gov/chesbay/RIMP/glossary.html#concentration Standard Methods For The Examination,of Water and Wastewater, 14th ed. 1975. Rand, M.C., WPCF, Chair, Joint Editorial Board. Published by APHA-AWWA-WPCF, Washington, DC. Page 4 of 10 v 2004 PHOSPHATE ANALYSIS PROTOCOL: This method follows the protocol for orthophosphate analysis published in: Standard Methods For The Examination,of Water and Wastewater, 14th ed. 1975. Rand, M.C., WPCF, Chair, Joint Editorial Board. Published by APHA-AWWA-WPCF, Washington, DC. Orthophosphates (PO43-) are used as fertilizers, and enter lakes and streams by storm run-off and melting snow. DO NOT REFERENCE THIS UNLESS YOU GO GET IT AND READ IT (AND YOU NEED NOT DO THAT!) In this experiment you will: 1. Obtain a water sample from the small pond behind the townhouses, or from a stream or river; 2. Treat the pond water with decolorizing charcoal, and filter it; 3. Make up several solutions containing known amounts of sodium hydrogen phosphate (Na2HPO4); 4. Obtain a “Quality Control” phosphate solution; 5. React all samples with molybdate-vanadate reagent and obtain absorbance readings at 400 nm. 6. Construct a standard curve of absorbance vs. [PO43-] using data from the known phosphate samples; 7. Determine [PO43-] in your pond water and “Quality Control” samples. You will need the following glassware and equipment: PHOSPHOROUS SET UP: [per pair unless noted otherwise] Sample bottle, acid washed, 2 dram (or 1 per student scintillation vial) 1 L bottle to make up 1 M HCl + stopper 1 [Under sinks in lab] Ehrlenmeyer flask, 125ml (for filtering 1 into) Volumetric Flask, 10.00 mL 7 Volumetric pipet, 1.00 mL 2 orange tape on one of the two Volumetric pipet, 2.00 mL 1 Volumetric pipet, 3.00 mL 1 Volumetric pipet, 5.00 mL 3 Red & blue tape on one of the 3 Blue pipet bulb/handle 2 Glass funnel, long stemmed 2 Filter paper, qualitative (to fit above) 2 boxes per riser Waste Jug (“Phosphorous”) 1 in Hood Ring Stand w/ 1 ring (to hold funnel) 1 Spatula 1 per charcoal set up Page 5 of 10 v 2004 You will need the following reagents: CHEMICAL PREP: 12 M HCl 83ml per pair of concentrated 12M HCL Molybdate-vanadate reagent 10 mL per pair: 0.02 M ammonium molybdate 1 set up per hood: (VI)/0.01 M ammonium 2x125ml flask w/ CC metavanadate in 4.0 M HCl* 10ml grad cyl. ORANGE Stock: 36.75ug/mL [PO4 ] 20 ml per pair 1 set up per hood 2x 250ml flask w/ CC 10ml grad cyl. RED Quality Control: 14.70 ug/ml 10 ml per pair 1 per hood 2 x Tall form beaker w/CC 10ml grad cyl. Decolorizing charcoal 2 50ml beaker: ½ full w/ a spatula Place on back bench on paper towels. Page 6 of 10 v 2004 STOCKROOM STAFF PREP DIRECTIONS: TO MAKE UP MOLYBDATE-VANADATE REAGENT: IN THE HOOD!!!!!!!!!!!! 1. DISSOLVE 25.0 G OF AMMONIUM MOLYBDATE (VI) [(NH4)6MO7O24.4H2O; MW 1235.86 G/MOL] IN 400 ML OF DISTILLED WATER IN AN ACID-WASHED 500 ML EHRLENMEYER FLASK. 2. DISSOLVE 1.25 G OF AMMONIUM METAVANADATE [NH4VO3, MW 116.98 G/MOL] BY HEATING TO BOILING IN 300 ML DISTILLED WATER, IN AN ACID-WASHED 1.00 L VOLUMETRIC FLASK. COOL TO RT 3. THEN, IN INCREMENTS WITH STIRRING CAREFULLY ADD 330 ML OF CONCENTRATED HCL TO THE ABOVE SOLUTION (#2). COOL. PLACE SET UP IN A BUCKET UNDER HOOD WHEN ADDING HCL. 4. POUR THE AMMONIUM MOLYBDATE [VI] SOLUTION INTO THE AMMONIUM VANADATE SOLUTION, AND DILUTE TO 1.00 L. 5. COLOR CODE: ORANGE ** TO MAKE UP STOCK PHOPSHATE SOLUTION: IN THE HOOD! 1. DISSOLVE 0.0534 G (NOTE EXACT MASS USED!!!!) OF SODIUM HYDROGEN PHOSPHATE [NA2HPO4.H20: MW: 137.99 G/MOL] IN 900 ML OF DISTILLED WATER IN AN ACID-WASHED 1.00 L VOLUMETRIC FLASK, AND THEN DILUTE TO 1.00 L. RECORD EXACT CONCENTRATION ON FLASK! 2. COLOR CODE: RED 3. MATH: MASS OF SOLID/ MW OF SOLID X 94,97G/MOL [MW OF PHOSPHATE ONLY]=_____PO4 G/LITER ________G/LITER X 106UG/G X 1L/1000ML =______UG/ML(LABEL AS…) *** TO MAKE QUALITY CONTROL SOLUTION: IN THE HOOD! 1. DISSOLVE .0214 GRAMS (NOTE EXACT MASS!!!!) OF SODIUM HYDROGEN PHOSPHATE [NA2HPO4.H20: MW: 137.99 G/MOL] IN 900 ML OF DISTILLED WATER IN AN ACID-WASHED 1.00 L VOLUMETRIC FLASK, AND THEN DILUTE TO 1.00 L. RECORD EXACT CONCENTRATION ON FLASK! 2. COLOR CODE: BLUE 3. MATH: SAME AS ABOVE Page 7 of 10 v 2004 PRELAB: A. OBTAIN A WATER SAMPLE. `You will need about 5 mL of water. Collect the water (fill the vial completely!) in the clean, acid- washed flask provided, seal it, and label it with your name, the date and time of collection, and the site of collection. Describe the exact time, location, and conditions under which you collected your sample. (This is part of your grade!) e.g. “The water sample I obtained was from the Winooski River delta at the Burlington Bike Path Bicycle Ferry landing. Due to severe drought (only 0.07 “ of rain in the last 30 days, according to www.NOAA.gov. ” Bring your sample and collection data to lab. I. Filtering and Decolorizing PROCEDURE: If your water sample has color to it, add a small spatula-tip full of activated charcoal to it – THINK TINY AMOUNT (about 20 mg. DO NOT WEIGH ON THE BALANCE). Swirl it around, let it sit for 5 min. and filter the sample through a moistened filter to remove the carbon. II. The Phosphate Assay To be done by TA: Make a stock solution of phosphate according to the procedure on page 5. Record the exact concentration on the container. To be done by the student: RECORD the exact concentration of the phosphate standard solution in your notebook. Before you come to lab: Calculate the volume of 12 M HCl you will need to dilute to 1 L to make a 1 M solution. ACID WASH ALL YOUR GLASSWARE WITH 1 M HCL, and then rinse with distilled water. Now that you have squeaky clean dishes: Making calibration standards & solutions for analysis: 1. Obtain 7 10.00 mL volumetric flasks. 2. Label the volumetric flasks "0" through "6". 3. Add 5 mL of deionized water to the "0" flask.. 4. To flask "1" carefully pipet in 1.00 mL of the stock phosphate solution using a volumetric pipet. 5. Following the chart on the following page, add 2.00 mL of the stock phosphate to flask 2, 3.00 mL of the stock phosphate to flask 3, and so on. To flask 5 add 5.00 mL of the Quality Control solution. To flask 6 add 5.00 mL of your water sample. 6. Add 1.00 mL of the vanadate-molybdate reagent to each flask, & dilute to the mark on the neck with deionized water. The bottom of the meniscus should be resting on the mark. Remember to thoroughly mix each flask. 7. Let the yellow color develop for 10 minutes or more. Read and record the absorbance of each sample at a wavelength of 400 nM. Page 8 of 10 v 2004 SET UP THE FOLLOWING CHART IN EXCEL BEFORE YOU COME TO LAB. PRINT OFF A HARD COPY AND BRING IT WITH YOU. Assay # mL stock PO43- Final volume [stock PO43-] A480 (mL) ug/mL 0 0 10.00 1 1.00 10.00 2 2.00 10.00 3 3.00 10.00 4 5.00 10.00 5 quality control 5.00 10.00 6 water sample 5.00 10.00 For best results, calibration standards should be prepared fresh each time a batch of samples is analyzed. Page 9 of 10 v 2004 Calculations: Calculate the [PO43-] in each of your standards. Generate a standard curve of absorption vs. concentration for your standards. Indicate what wavelength you used. Calculate the best fit straight line for your data. See chapter 4 in the Excel Manual to find out how to do this, at http://academics.smcvt.edu/chemistry/CHEM 103/week_2.htm Using your standard curve best fit line, calculate the [PO43-] in your water sample. Also calculate [PO43-] in your quality control. Calculate a % absolute error between what you KNOW your quality control [PO43-] content is and your experimental value. WASH & RETURN ALL GLASSWARE! Slope of best fit straight line= 4.111 x 10-6 Y –int = 2.63 x 10-3 400 nm The equation of the best fit straight line is: Y = mx + b, where Y = absorbance, m = slope, x = [PO4-3], and b = Y-intercept. Thus, if your water sample has an absorbance of 0.0151 at 400 nm, the [PO43-] is: 0.0151 = 4.111 x 10-6([PO43-]) + 2.63 x 10-3 [PO43-] = (0.0151 – 2.63 x 10-3)/ 4.111 x 10-6 = 3030 ug/L Page 10 of 10 v 2004 You originally used 5.00 mL of water (0.005 L) and diluted it to 10.00 mL (0.0100 L). Thus, the original [PO43-] in your sample was: (3030 ug/L )(0.0100 L/0.005 L) = 6060 ug/L You can use this as a suggested format to set up your data in Excel…Notice that this data is different than yours! ALL WASTE FROM THIS EXPERIMENT GOES IN THE JUG LABELLED “PHOSPHATE WASTE” IN THE HOOD.