Write your own lab Spectrophotometry/Colorimetry Sheila MacIntyre University of Pennsylvania Spectroscopy Nitrate Concentration Determination Teacher Section: Background Information : In this lab students will determine the concentration of nitrates in local water sources using a spectrophotomer. Students will perform a serial dilution from a standard and graph the absorbance vs. concentration using the Beer-Lambert Law and from this graph they will determine the concentration of nitrate in their water samples. This lab is designed to follow an environmental science unit on water quality concentrating on the importance of nutrient cycling in an ecosystem. In this lab, students will determine the - concentration of nitrate ions, NO 3 , in fresh water samples from a pond, canal and the Delaware River which is located near the school. The concentration of nitrate will be expressed throughout this section in units of mg/L of nitrogen that is in the form of nitrate. Nitrates are an important source of nitrogen necessary for plants and animal to synthesize amino acids and proteins. Most nitrogen is found on the earth in the form of nitrogen gas, N 2. The nitrogen cycle converts nitrogen into forms that are usable by plants and animals. Formation of - nitrate in freshwater systems results from ammonia is converted to nitrite (NO 2 ) by nitrosomona bacteria and nitrobacter quickly converts nitrite to nitrate. Legumes are plants that are able to undergo nitrogen fixation in the root nodules. Plants and animal decomposition, animal excretion and fertilizer production are other ways nitrogen is recycled in the environment. Nitrate ions are found in freshwater systems as a result of these natural and manmade sources. High nitrate concentrations contribute to a condition in lakes and ponds called eutrophication, which is an excessive growth of aquatic plants and algae. This causes reduced clarity, unpleasant odors and often algal blooms and fish kills. An accumulation of dead biomass accumulates on the bottom of the lake or pond and causing problems in the natural recycling of nutrients. Nitrate pollution of surface and ground water has become a major ecological problem in agricultural areas from fertilizer runoff. High nitrate levels in drinking water can cause a fatal disease in infants called methemoglobinemia, or Blue Baby Syndrome. Other sources of nitrogen pollution are untreated waste water, urban runoff, automobile and industrial emissions and live stock wastes. Prerequisite knowledge : Point and non point pollution sources Freshwater quality parameters Nutrient loading-Nitrogen Cycling Eutrophication Write your own lab Spectrophotometry/Colorimetry Assessment : Lab Report Introduction Section (Question, background research) Hypothesis Procedure (materials and procedures) Data/Results (tables, graphs and calculations) Discussion (sources of error) Conclusion (relate to hypothesis) Equipment/Materials : It is important that the sample be taken below the surface of the water and away from the shore. Equipment such as boots or long rods for water collection are options, a 100 ml sample is required. Keep sample refrigerated or in an ice chest, if the testing is not within a few hours. Safety Alert: Cadmium metal is used as the nitrate reducing agent: Carcinogenic, dust or fume inhalation is toxic. Hazard Code B-Hazardous Dispose cadmium waste according to your state a local regulations. Reagents may be purchased from LaMotte Company, www.lamotte.com Mixed Acid Reagent (V-6278-H) Nitrate Reducing Agent (V-6279-C) Nitrate-N Kit- contains both reagents (3649-SC) Recommended wavelength is 540nm for a spectrophotometer. Preparation of Stock Solution - Prepare the nitrate standard solution with a concentration of 2.5 mg/L NO 3 - N: For a 100mg/L standard add 0.607 g of NaNO 3 to make a 1 liter of solution. Dilute 100mL of this solution in 900 mL of distilled water to make a 10mg/L solution. Combine 250mL of the 10 mg/L - solution with 750mL of distilled water to make a 2.5 mg/L NO 3 -N concentration. Time requirements Water sampling by students may be incorporated into a field trip or the instructor can collect the samples before the class period. A 90 minute class period (not including water sampling) and appropriate time for the lab write up. Write your own lab Spectrophotometry/Colorimetry Student Section: Learning Objectives: SWBAT: • Perform water collection techniques • Perform a serial dilution of a standard stock solution. • Create an absorbance vs. concentration graph based on the Beer-Lambert Law • Properly use a spectrophotometer/calorimeter for data collection • Obtaining the concentration of nitrate in fresh water systems from an Excel generated graph Handouts/Instructions : Materials 2 100mL beakers Wash bottle 7 Erlenmeyer flasks and 7 rubber Two graduated cylinders stoppers Tissues Mixed Acid Reagent 0.1 g measuring spoon Nitrate Reducing Reagent Gloves Nitrate Standard Safety goggles Two cuvettes Lab Procedure has been adapted from Vernier Water Quality: Part 1 -Dilution – 1. Add about 30 mL of 2.5 mg/L NO 3 -N standard solution to a 100 mL beaker. 2. Obtain about 30mL of distilled water in another 100 mL beaker. 3. Label four clean, dry, Erlenmeyer flasks 1 – 4. – 4. Pipette 4, 6, 8 and 10 mL of 2.5 mg/L NO 3 -N solution into Flasks 1 – 4, respectively. 5. With a second pipette, deliver 6, 4, and 2 mL of distilled water into Flasks 1 – 3, respectively. (Flask 4 has no distilled water added to it.) 6. Thoroughly mix each solution with a stirring rod. Clean and dry the stirring rod between stirrings. 7. Measure 5 mL of the standard from Flask 1 into a graduated cylinder. Discard the solution remaining in the flask as directed by your instructor. Part 2: Preparing your Standards (oxidizing agent is added to give the sample a color) CAUTION: CADMIUM IS A TOXIC METAL-USE GLOVES AND GOGGLES AND DISPOSE OF SOLUTIONS IN PROPER CONTAINER 8. Add 5 mL of Mixed Acid Reagent to the graduated cylinder containing the standard from Flask 1, to bring the volume to a total of 10 mL. 9. Pour the contents of the graduated cylinder back into the flask. Stopper each flask and shake. 10. Repeat Steps 3 – 5 for each of the remaining standards. 11. Use the 0.1 g plastic spoon to add two spoonfuls (~0.2 g) of Nitrate Reducing Reagent to each of the flasks. Write your own lab Spectrophotometry/Colorimetry 12. Stopper the flasks and invert at a rate of 50 – 60 times per minute for 2 minutes. Wait 12 minutes for a complete reaction and best test results. During the 12 minute reaction period, proceed to Step 14 to continue with lab preparation. 13. Note: Any undissolved portion of Nitrate Reducing Agent that remains in the bottom of the tube will not adversely affect results. Part 3: Setting up the Spectrophotometer While you are waiting for the color to develop, determine the λmax by using two spectrophotometer cuvettes. 14. Prepare a blank by filling an empty cuvette ¾ full with distilled water. Seal the cuvette with a lid. To correctly use a cuvette, remember: o All cuvettes should be wiped clean and dry on the outside with a tissue. o Handle cuvettes only by the top edge o All solutions should be free of bubbles. o Always position the cuvette with its reference mark facing toward the white reference mark at the top of the cuvette slot on the spectrophotometer. 15. Prepare a sample, as instructed in step 14 using the most concentrated solution. 16. Set the wavelength to 400nm and set to display absorbance. Use the blank to set the zero and then place your sample in and record absorbance. 17. Remove the sample and replace the blank. 18. Change the wavelength to 425 and repeat steps 15-17. 19. Continue this process by changing the wavelength by 25nm increments, until you have the correct wavelength range of the spectrophotometer. Chose the λmax and set the spectrophotometer to that wavelength to collect the data for the four nitrate standard solutions. Part 4: Creating a standard curve This process will create a standard curve that will be used to determine the nitrate concentrations of the samples. Your spectrophotometer should be set at 540nm if you did the process correctly. 20. Empty the water from the cuvette. Using the solution in Flask 1, rinse the cuvette twice with ~1 mL amounts and then fill it ¾ full. Wipe the outside with a tissue and place it in the spectrophotometer. After closing the lid, wait for the absorbance value to display. Record. 21. Discard the cuvette contents as directed by your teacher. Using the solution in Flask 2, Repeat step 20 for all Flasks. Record your data. 22. Create a graph of absorbance vs. concentration on the graph paper or a computer. 23. Examine the graph of absorbance vs. concentration. To see if the curve represents a direct relationship between these two variables. Determine the best-fit linear regression line will be shown for your data points. This line should pass near or through the data points and the origin of the graph. 24. Show instructor your graph before you proceed. Part 5: Preparation of water samples 25. Prepare the water samples for testing. 26. Add 5ml of sample 1 to a graduated cylinder. Write your own lab Spectrophotometry/Colorimetry 27. Add 5 mL of Mixed Acid Reagent to the graduated cylinder containing sample 1 and bring the volume to a total of 10 mL. 28. Pour the contents of the graduated cylinder back into the flask. Stopper each flask and shake. 29. Repeat Steps 25-28 for each of the samples. 30. Use the 0.1 g plastic spoon to add two spoonfuls (~0.2 g) of Nitrate Reducing Reagent to each of the flasks.