1 Measuring ph with Smart Cuvettes Hardware Setup for ph Measurements ph is measured in Absorbance mode. Configure your spectroscopy components for Absorbance measurements. Sample Absorbance setups for the STS and Spark are shown below: STS/ecoVis setup for measuring ph in Absorbance mode Spark setup for measuring ph in Absorbance mode
2 Measuring Absorbance Spectra to Determine ph 1. Configure your spectroscopy components for Absorbance as shown above. Connect the spectrometer to your computer using the USB cable and turn on your light source. Allow the spectrometer and light source to warm up for at least 15 minutes. 2. Start the OceanView software. Click the Start button, then select All Programs Ocean Optics OceanView OceanView or use the Desktop shortcut created when you installed the software. 3. Select the Spectroscopy Application Wizards option on the Welcome Screen. 4. Select the Absorbance (Concentration) option in the Spectroscopy Wizards window to start the Absorbance Wizard.
3 5. Select the Absorbance only option. 6. Place the Smart ph Cuvette into the cuvette holder ensuring that the ph patch applied to the window of the cuvette is in the light path for the measurement. NOTE: The ph patch installed in the cuvette is a transmissive ph patch. This means that light must pass through the patch for the measurement so the patch should be placed in the path between the light source and spectrometer for a setup with direct attach accessories (e.g., Spark) or between the light source and fiber for a setup with a fiber connecting the spectrometer to cuvette holder (e.g., STS). 7. Use a transfer pipet to fill the cuvette ~⅔ full with an acid (low ph) sample (e.g., white vinegar) for your reference measurement. NOTE: The ph of several household solutions that can be used as acid and base samples for these measurements is shown in the chart at the end of the procedure. 8. Set your acquisition parameters in the Set Acquisition Parameters window. a. Click the Automatic button to automatically adjust the Integration Time to the optimum value.
4 b. Set Scans to Average to 20 and Boxcar Width to 10 to reduce measurement noise and improve the measurement. You can set Scans to Average to a higher value but this will slow down your acquisitions (measurement time equals the number of averages times the integration time). c. Place a checkmark in the Nonlinearity Correction box if this feature is available for your spectrometer. d. Click Next. 9. Click the Store Reference (yellow light bulb) to store a reference spectrum. Click Next. 10. Block light from entering the spectrometer using the dark cuvette accessory (EDU-CUV- DARK). Do not turn your light source off. Click the Store Background (gray light bulb) button to store a background spectrum. 11. Remove the dark cuvette accessory from the cuvette holder and click Finish. 12. Place the cuvette containing the acid reference sample back in the cuvette holder in the same orientation used in Step 6. Your Absorbance spectra will be displayed in the AbsorbanceView window. You can adjust the graph appearance using the graph tools above the graph. For example, click the Scale Graph Height to Fill Window button to zoom in on the spectrum. 13. Remove the reference sample (Sample 1/acid sample) from the cuvette using a pipette and use a new pipette to fill the cuvette ~⅔ full with a base (high ph) sample (e.g., soap solution, ammonia). Allow the sample to sit for 5 seconds, use the pipette to remove the base sample from the cuvette and fill the cuvette ~⅔ full with fresh base sample. NOTE: The ph of several household solutions that can be used as acid and base samples for these measurements is shown in the chart at the end of the procedure.
5 14. Wait 30-60 seconds for the sample and ph patch to reach equilibrium and save the absorbance spectrum (e.g., Sample 2) by clicking the Configure Graph Saving button configure the File Writer. to a. Select the directory where you want to save the file. b. Enter a filename for your spectrum (e.g., Sample 2). c. Click Apply and then Exit to close the dialog box. 15. Click the Save Graph to Files button to save your absorbance spectrum. 16. Remove the base sample from the cuvette with a pipette and use a new pipette to fill the cuvette ~⅔ full with the sample you want to measure ph for (Sample 3/analytical sample). Allow the sample to sit for 5 seconds, use the pipette to remove the analytical sample and fill the cuvette ~⅔ full with fresh sample. 17. Wait 30-60 seconds for the sample and ph patch to reach equilibrium and save the absorbance spectrum (e.g., Sample 3) as described in Steps 14-15. 18. Repeat Steps 16-17 to measure and save absorbance spectra for additional analytical samples.
6 Using the Excel Worksheet to Calculate ph from the Absorbance Spectra NOTE: The Excel file for calculating ph from absorbance spectra saved from OceanView can be downloaded from the STS Education Kit Resource Page or the Spark Education Kit Resource Page under the Sample ph Experiments section. 1. Open the Excel file ph Kit Worksheet and then open each of the absorbance spectra saved above. 2. In the tab called Raw Data Input, copy and paste the saved absorbance files as described below: a. Copy all data in columns A and B from the base file and paste into cell A1. b. Copy all data in columns A and B from the first analytical file and paste into cell F1.
7 3. Scroll to columns M O and enter the following values for the standard Bromocresol green Smart ph cuvettes: a. Analytical Peak: 620 b. Baseline 1: 510 c. Baseline 2: 750 4. The graph below these values should show absorbance curves that look similar to these: 5. Click on the Analysis tab. In the yellow highlighted fields below the graphs, input the estimated ph values of the acid (Sample 1) and base (Sample 2) samples you measured with the Smart ph cuvette. NOTE: The estimated ph for various household samples can be found in the chart at the end of the procedure or with a Web search.
8 6. The calculated ph value of the analytical sample will be shown in the orange highlighted box (Sample 3). 7. Determine the ph for other samples by pasting the absorbance spectra for the samples into cell F1 as described in Step 2.
9 Household Samples for ph Measurements with Estimated ph http://www.compoundchem.com/2015/07/09/ph-scale/