Determination of Sodium using Atomic Emission 1. Purpose The purpose of this procedure is to determine the concentration of sodium ion in parts per million in an unknown sample. 2. Background Atomic emission (AE) spectroscopy is a very well established technique in analytical chemistry, with applications in many fields of science. It has been used in the pharmaceutical industry to determine the amount of calcium in antacid tablets. Marine biologists have used AE to determine mercury levels in ocean sediment and fish tissue. In one specific example from the literature, environmental scientists at a wildlife research center used AE coupled to gas chromatography to determine phosphorous concentrations in duck gizzards. 2 Like all spectroscopic methods, AE use electromagnetic energy to determine the concentration and/or identity of some analyte(s). In atomic spectroscopy, the elements present in a sample are converted to gaseous atoms by an atomizer. In AE, the flame atomizer also serves to produce an electronically excited species of the analyte. These excited species emit radiation upon relaxation, producing the analytical signal at a specific wavelength. In this laboratory, sodium is the analyte of interest and an acetylene flame provides the energy for excitation. The sodium atoms are thermally excited and emit radiation with a wavelength of 589 nm. The intensity of the radiation is proportional to the concentration of the sample. This relationship allows an analyst to construct a calibration curve with standards of known concentration. Using least-squares analysis, the concentration of an unknown sample can be determined from a single emission intensity measurement. 3. Materials and Equipment 1 L volumetric flask funnel 500 ml volumetric flask sodium chloride 100 ml volumetric flask (6) 1 L plastic bottle Spatula 500 ml plastic bottle 10 ml graduated cylinder 100 ml plastic bottle (7) 100 ml graduated cylinder nanopure water Eppendorf micro-pipette (1000 µl) and disposable tips Varian SpectrAA 200 Flame Atomic Absorption Spectrophotometer 4. Safety / Special Handling Procedures Protective eyewear must be worn at all times. Acetylene is run into the AE from a gas cylinder. If you need help opening the main valve, please ask your instructor. Many internal parts of the AE are very hot. Ask your instructor for assistance if you need to open the cover of the AE. Notify the instructor if there is a problem with the instrument. For more general safety in the laboratory, please refer the appendix. AE Page 1
5. Experimental Method 5.1. Preparation the Stock Standard Calculate the amount of pure sodium chloride required to give one liter of approximately 100- ppm sodium. Weigh out the NaCl and record the actual, exact mass used. Quantitatively transfer the NaCl to a one liter volumetric flask using a funnel and a wash bottle. Transfer the solution to a plastic bottle and label it "Sodium Stock Standard" and calculate/record the exact concentration 5.2. Preparation the Unknown Obtain an unknown from the instructor and quantitatively transfer the unknown to a half-liter volumetric flask with a water bottle. Dilute to the mark with nanopure water, mix and transfer to a plastic bottle. Label it "Unknown Sodium". 5.3. Preparation of the Working Standards 5.3.1. Obtain a 1000 µl Eppendorf micro-pipette fitted with a proper size plastic disposable tip. If you need a review on the proper use of micro-pipettes, please let your instructor know or consult the Appendix A at the end of this procedure. Before start using the micro-pipette, you should verify and calibrate if the pipette is working properly (see Appendix A for more on this). Calculate the volume (in µl) of stock standard sodium necessary to give 100 ml of around 0.2 ppm Na as close as possible based upon your stock solution concentration. The aliquot size should be a nice round number. In other words, if your stock solution concentration is 127 ppm Na, then, you will likely be making 0.0217 ppm Na solution as opposed to 0.0200 ppm solution. 5.3.2. Transfer an aliquot of this volume to a 100 ml volumetric flask, dilute to the mark, mix and (if you need to reuse your volumetric flask) transfer to a small plastic bottle labeled "AE #1". This is the lowest concentration standard for your calibration curve. 5.3.3. Continue to prepare the standards as in step 5.3.1 until 6 total solutions have been prepared. The concentration of each standard should increase by 0.2 ppm. For example, the concentration of Solution 2 should be about 0.4 ppm and the concentration of Solution 6 should be about 1.2 ppm. You should tabulate the concentrations and volumes used to make up your standard solutions; perform at least one sample calculation in your notebook. 5.4. Preparation of a "top standard" Sodium Solution Per step 5.3, the most concentrated standard solution is approximately 1.2 ppm Na +. Next, prepare a sodium solution which is more concentrated than the most concentrated solution in your set. This solution is not a true standard, but will be used to optimize the instrument response (photomultiplier tube signal). Therefore, its exact concentration does not need to be known and it does not need to be made as accurately as the standards. Prepare around 100 ml of approximately 5 ppm Na + from the concentrated stock solution. You may use a graduated cylinder and beaker for this solution preparation. Label this solution top standard. AE Page 2
5.5. Analysis using the Varian SpectrAA 200 spectrometer 5.5.1. Bring the following to the instrument room: 1. All 6 standard solutions 2. Unknown sodium solution 3. 400 ml beaker with nanopure water 4. Top standard 5. Notebook 6. Kim Wipes 5.5.2. On the front of the instrument is a tray for the samples. There is a plastic capillary tube that comes out of the instrument onto this tray. Place the beaker of water on the left side and the top solution on the right. The space between the two will be for the various samples. Refer to Figure 1 for a picture of the instrument. Note that in this lab the Atomic Emission experiment uses the same apparatus and fuel mixture as the Atomic Absorption experiment (acetylene and air). Figure 1: Varian SpectrAA 200 Atomic Absorption Spectrophotometer 5.5.3. Turn on the acetylene gas. The acetylene gas cylinder is located to the right of the instrument. Turn the main valve at the top of the cylinder counter-clockwise so that it is completely open. Do not touch any other knobs on the regulator! See Figure 2 for a picture of the cylinder and regulator. Ask your instructor for help if you have any questions. 5.5.4. Turn on the compressed air. The compressed air is from an in-house source and the valve is located on the left side of the (old) fume hood next to the gas chromatograph (GC) on the south side of the room. Open the valve by turning the handle 90 away from you. (Figure 3). AE Page 3
Figure 2: Acetylene gas cylinder and regulator 5.5.5. Compressed air valve Turn on the computer, if it is not already running. Start Varian SpectrAA 100/200 application upon startup. See Figure 4 for a screenshot of the application. Figure 4: 5.5.6. Figure 3: Varian SpectrAA application Turn on the instrument by flipping the switch on the lower left corner of the instrument (Figure 5). Once you have turned on the instrument, you will get a warning on the computer. Ignore the message and click "OK". Figure 5: Varian SpectrAA Power switch and the Ignite button 5.5.7. Ignite the flame by the following procedure. 5.5.7.1. First, place the capillary tube into the beaker containing nanopure water. Switch the gas control knob (located front right of the instrument see Figure 6) from "OFF" to "AIR" setting. As the compressed air exits the burner head, it will make a hissing sound as soon as you switch the knob to air setting. AE Page 4
Instrumental Analysis Laboratory Safety Rules A. Instructions: Carry out all manipulations in accordance with instructions and the safety rules and procedures given herein. B. Eye Protection: All students and staff working in the laboratory must wear safety glasses at all times. If a student needs to be reminded more than three times to wear goggles, she/he will be dismissed from lab for the remainder of the day, and will not be given an opportunity to make up the work. C. Apparel: The clothes you wear in lab are an important part of your safety equipment, and should offer protection from splashes/spills. Closed toed shoes (sneakers are fine), Full-length pants or a full-length skirt, and A shirt that completely covers your torso (i.e. at minimum, a t-shirt). In other words, you must NOT wear shorts to lab. You must NOT wear flip-flops, sandals, or crocs. You must NOT wear tank tops, halter tops, spaghetti-strap tops, or low cut jeans to lab. Exposed abdomens, hips, and backs are not safe in the lab. D. Gloves: Gloves are an important part of personal protection. Gloves will be available at all times in the laboratory. Your instructor will require their use when appropriate. E. Food: Food, drinks, and gum are not allowed in lab. None at all, not even water bottles. F. Sanitation Issues: Be sure to wash your hands before leaving lab, before you eat anything outside of lab, and before you answer your cell phone. G. Music: Individual headphones are not allowed. Your may choose to play music for the entire class. H. Cell Phones and Other Electronic Devices: Cellular phones and other electronic devices that you do not need to perform your laboratory work should be put away. I. Other: All students are explicitly prohibited from: 1. conducting any unauthorized experiments. 2. removing chemicals or apparatus from the laboratory for any reason. 3. working in the lab alone, or at other than regularly scheduled lab periods. 4. smoking in the laboratory or within 20 feet of any doorway. 5. impeding movement in aisles or through doorways with bags, skateboards, etc.
Figure 6: Gas control Figure 7: capillary tube 5.5.7.2. Next, slowly turn the acetylene flow knob (Figure 6) counter-clockwise. Simultaneously, press the ignite button (Figure 5) located next to the main power switch to ignite the flame on the burner. Once the flame has been ignited, adjust the acetylene flow so that it is sufficient to maintain the flame. The flame is visible through the window above the sample introduction port (Figure 7). It should appear green in color through the protective shield for the burner. 5.5.8. Once the flame has been lit, ensure that the capillary tube is still in the beaker containing nanopure water and that it contains sufficient water. This is the blank (Figure 7). 5.5.9. Next, on the computer, click the "Worksheet" button on SpectrAA 100/200 application (Figure 8). Figure 8: Computer Screenshot 5.5.10. Click "New From Template" button. You should see a window with the available template. Choose "sodium by AE" and click "OK" (Figures 9 and 10). AE Page 5
Figure 9: Load Worksheet "New From Template" Figure 10: Select "sodium by AE" 5.5.11. Next, you will be prompted to name your worksheet. Enter the name of the file in the Name box. Record this name in your lab notebook. Enter your name in Analyst box and click "OK" (Figures 11 and 12). Figure 11 Figure 12: Identify your worksheet 5.5.12. At this point, the computer should show the screen in Figure 13. Figure 13: Instrument control 5.5.13. You are ready to begin the analysis. Click the "Read" button on the left side to start. You should see a dialog box named "Analysis Checklist". Verify that these settings are correct and document them in your notebook. Ask your instructor for help checking the slit width. Click "OK" to continue. Wait for a few seconds while the instrument makes the necessary adjustments. AE Page 6
5.5.14. Once the instrument is ready, it will prompt you to present your top standard sodium solution. Place the capillary in the top standard solution and click "OK". The instrument will take the reading of your top standard. Remember to wipe off the capillary with a Kim wipe between all readings. 5.5.15. When the instrument has performed the reading from the top standard, it will prompt you to remove the top standard. Replace the top standard with the blank. Once you have the blank in place, click "OK" to continue. 5.5.16. Next, the instrument will ask for sample #1. Remove the blank, place the capillary in sample #1 and click "OK". The instrument will acquire emission data from the sample solution for approximately 15 seconds. You can watch the progress at the top right of the program screen. Do not remove the capillary tubing until the data acquisition is complete. Once the reading is done for 15 seconds, place the capillary back in the beaker containing nanopure water. 5.5.17. After the instrument has taken the measurement for sample #1, on the computer move the cursor to the cell for sample #2 and put the capillary in sample #2. Click "Read" button and repeat the process for sample #2. It is important that the cursor be on the cell of the sample to be read. Otherwise, the data will not be recorded with the correct sample. 5.5.18. Continue the process in step 5.5.1.7 for all of the standard solutions and the unknown (which should be sample #7). Set up a table and record the emission values in your notebook. 5.5.19. When all of the solutions have been read, click the "Stop" button to stop analysis. Ensure that all of the emission values are documented in your notebook. It is important that both your standards and samples are analyzed on the same day. The instrument response can change day to day. Running the samples and standards at the same time gives the most accurate results. 5.5.20. Print out the data by the following procedure. Exit the worksheet: Go to the "Exit" menu and select "Return to Main Index". In the Main Index, click "Report" button. Refer to Figure 8 for a screen shot of the Main Index. Figure 14: Select your worksheet Figure 15: Print your report AE Page 7
5.5.21. Select your report file (Figure 14). Click the "4. Report" tab. Click "Print Preview " button to preview the report (Figure 15). If the preview looks satisfactory, print your result by clicking the "Print " button on the right-hand side. When your data has printed, click the close button. Double-check that the data print-out and the values in your notebook agree. 5.5.22. Exit SpectrAA application (Figure 8). 5.5.23. Shut off the acetylene gas: Turn the acetylene knob on the instrument clockwise. Close the main valve on the acetylene tank (Figure 2). 5.5.24. Turn off the compressed air: Switch the gas control knob on the instrument to the off position (Figure 6). Turn off the in-house compressed air (Figure 3). 5.5.25. Turn off the instrument power (Figure 5). AE Page 8
Data Analysis / Calculations 5.6. Enter your data into the least squares program on the computer in the balance room or the Chemistry Computer Lab. If you need help with this program, ask your instructor. (You can also make your own Excel table and graph, if you prefer.) Enter the X value data as the concentration of the linear standards and the Y data as corresponding emission values. Enter the emission value of the unknown and record the concentration value in your notebook. Print out this data and turn it in with your notebook pages. Remember to write your name and date on any printouts. All graphs should have the x and y axes properly labeled and should have a title. You may write or type these into your graph. 6. Reporting Requirements Report the concentration (in ppm) of sodium in the 500 ml unknown solution to three decimal places. 7. Waste Disposal Discard all solutions down the drain. 8. References 1. Skoog, Fundamentals of Analytical Chemistry, 8 th Edition, Chapter 28 (atomic spectroscopy) and Chapter 8 section C (calibration curves). 2. Johnston, J.J. et. al. Environ. Sci. Technol., 2000, 34, p. 1856 1861. AE Page 9
Appendix A: Operation of Eppendorf Adjustable Pipettes A1. Volume Setting The volume is adjusted by pressing down the lateral catch and turning the control button at the same time. It is advisable to carry out volume setting from the higher down to the lower value (i.e. first go above the desired volume and then return to the lower value). A2. Pipette tips Typically the color of the control button will correspond to the color of the eppendorf tip or tip rack. For the best precision and accuracy, pre-wet all new tips by aspirating and dispensing liquid 2-3 times before pipetting. A3. Aspirating liquid! Attach suitable pipette tip to the pipette firmly.! Press down the control button to the first stop (measuring stroke).! Immerse the pipette tip vertically ~3 mm into the liquid.! Allow the control button to slide back slowly.! Pull the tip out of the liquid slowly.! To remove any remaining droplets, dab with non-fibrous cellulose material, ensuring that liquid does not come out of the tip. You can also dab on the side of the beaker containing the liquid you are pipetting. A4. Dispensing liquid! Hold the tip at an angle against the inside wall of the tube/flask.! Press down the control button slowly to the first stop (measuring stroke) and wait until the liquid stops flowing.! Press down the control button to the second stop (blow-out) until the tip is completely empty.! Hold down the control button and pull the tip out of the inner wall of the tube/flask.! Allow the control button to slide back slowly.! Tip is ejected by pressing the control button to the final stop. Do not lay down the pipette when a filled pipette tip is attached as this may result in liquid entering the pipette. A5. Verification of pipette You can verify that the pipette is performing accurately by dispensing nanopure water from a pre-wetted tip into a tared flask or tube onto an analytical balance. Typically, for this experiment, test at 500 µl for the 1000 µl pipette. You should do so BEFORE you start using the pipette. Convert the mass to volume by dividing by the density at room temperature. For example, the density of water is 0.9982 mg/µl at 20 C. This number is the volume actually delivered by the pipette. Determine the error relative to the set value. Repeat a few times to verify that the pipette is accurately delivering water. If not, consult your instructor. AE Page 10