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Lab 5: Spectrophotometry Learning Outcomes Addressed in this Lab: LO-2: Explain the methods of inquiry used by scientists. LO-10: Demonstrate proficiency in performing basic measurements and metric conversions utilized in the sciences. LO-11: Apply scientific reasoning to investigate question...

Lab 5: Spectrophotometry Learning Outcomes Addressed in this Lab: LO-2: Explain the methods of inquiry used by scientists. LO-10: Demonstrate proficiency in performing basic measurements and metric conversions utilized in the sciences. LO-11: Apply scientific reasoning to investigate questions and utilize scientific tools such as microscopes and laboratory equipment to collect and analyze data. LO-12. Use critical thinking and scientific problem-solving to make informed decisions in the laboratory. Objectives  Practice measurement techniques using glass pipettes, micropipettes and graduated cylinders  Calculate proper amounts of solute and solvent solution required to produce percentage volume per volume (% v/v) solutions.  Describe how spectrophotometry uses light to measure concentration in solutions  Practice operation of a spectrophotometer and analysis of data resulting from that operation. REMINDER: Don’t forget to read this entire lab and complete the pre-lab assignment BEFORE the start of lab (as directed by your instructor either on paper or online). Background Information Biologists routinely determine the presence and measure the concentration of dissolved chemicals using a spectrophotometer. Spectrophotometry is based on the principle that atoms, molecules, or even chemical bonds, absorb a unique pattern of wavelength of light. Fig. 5-1 shows the different wavelengths of electromagnetic radiation. Notice that in the visible spectrum, different wavelengths have different energies and different color. Fig. 5-1 Light as a wave of various wavelengths. In the visible spectrum, the wavelengths are interpreted by cells in our eyes as different colors. License CC BY SA 4.0 (Credit: Horst Frank) A colored solution, such as an oxidized guaiacol solution, appears that way because some of the light entering the solution is absorbed by the colored substance. A clear solution will allow almost all the light to pass through. The absorbance (A) (amount of light absorbed by the solution) can be determined by using the spectrophotometer. The spectrophotometer quantitatively measures what fraction of the light passes through a given solution and indicates on the absorbance scale the amount of light absorbed compared to that absorbed by a clear solution. The darker the solution, the greater its absorbance. BIOL-1406 Lab Manual Page 45 of 118 5. Ejecting the tip. Hold the pipette shaft over a suitable disposal container and press the tip ejection key to discard the tip. For this activity, deliver your 500 µl of blue dye onto the provided piece of waxy parafilm paper. Place each team members drop away from the others in a line so that you can compare volumes. At the end, observe the droplets – they should all be the same size since you measured the same volume. How did your team do with the 500 µl droplets? Great job! Now repeat this activity after changing the volume on the micropipette to 150 µl and place these droplets next to your set of 500 µl drops – they should be much smaller and again should match between team members. Notice how much less distance the button moves before the first stop when the volume we’re measuring is lower. How did your team do with the 150 µl droplets? Activity 5-2 Blue-dye Simple Dilution Dilutions are commonly used in biology labs for a variety of purposes. Therefore, it is important that you understand how to perform simple dilutions and be able to determine what dilution has been produced. In the meantime, you need to learn how to select appropriate pipettes and read the volume of pipettes accurately. A. Simple dilution and dilution calculations. In simple dilution, a specific volume of relatively concentrated solute solution (often called the stock solution) is added to an appropriate volume of a solvent to achieve a desired concentration. The diluted material must then be completely mixed to produce a uniform solution. For example, a 1:5 dilution means you combine 1 unit volume of the solute solution with 4 units volume of the solvent. If you need 100 ml of this 1:5 diluted solution, you would need to mix 20 ml of the solute solution with 80 ml of the solvent. Dilution fold = amount of stock solution : total final volume In the above example, dilution fold = 20 ml : (80+20) ml = 20 ml: 100 ml = 1:5 Another example: adding 0.5 ml of a blue-dye solution to 9.5 ml of water gives rise to a dilution fold of 1:20 because dilution fold = 0.5 ml : (9.5+0.5) ml = 0.5 ml : 10.0 ml = 1: 20. Now it is your turn to solve the following cases of simple dilution calculation: Case #1: adding 1 ml of concentrated solution to 99 ml of solvent makes the dilution fold of _________________________. BIOL-1406 Lab Manual Page 48 of 118 Activity 5-3 Blue-dye absorbance measurement Now that we have learned how to use the micropipettes and glass pipettes for proper measurement and prepared our blue dye samples, let’s practice using a spectrophotometer to confirm that we correctly calculated and prepared out dilutions. I. Calibrating the Vernier SpectroVIS Spectrophotometer 1. There is no on/off switch for the spectrophotometer; just make sure that the USB cable is connected to the computer. 2. Double click on the LoggerPro 3.8.2 icon on the computer desktop. 3. Fill a cuvette (about ¾ full) with the blank solution (water for the blue dye experiment). 4. Insert the cuvette in the sample chamber, making sure that the clear side of the cuvette is in the path of light (facing the arrow sign ►). 5. On the top menu, click on Experiment. Scroll down to “Calibrate” and select “Spectrometer:1”. 6. Wait 60 seconds for the lamp to warm up. 7. Click on “Finish Calibration” and on “OK”. The spectrophotometer is now calibrated. II. Sample Preparation We’ll be using the 3 samples prepared in the previous activity. Make sure you have mixed each solution with the Vortex mixer before using. III. Setting the spectrophotometer for data capture and determining the wavelength of maximum absorbance for the blue dye solution 1. On the top bar, find the “Configure Spectrophotometer” icon. 2. Select “Absorbance vs. Wavelength” as the Collection Mode. 3. Click OK. A graph should appear with wavelength (nm) on the x-axis and absorbance on the y- axis. 4. Fill a cuvette about ¾ full with the solution containing the blue dye solution in test tube 1. 5. Click “Collect” and then “STOP.” Do NOT discard this solution – set the cuvette aside and we’ll use it again in the next activity. 6. The full chromatogram of the blue dye should be displayed. Click on Autoformat if necessary. What is the wavelength showing the highest level (maximum) absorbance for the blue dye? ____________________ IV. Confirming Blue Dye Dilution Concentrations 1. On the top bar, two places left of the ‘Collect’ icon, click ‘Configure Spectrophotometer” 2. This time, select “Absorbance vs. Concentration” as the Collection Mode. Type-in “%” as the unit of concentration. Leave everything else as is. 3. Under the column showing a list of wavelengths, click on “Clear Selection” and then select the wavelength closest to the one that you have found in section III. Click OK. 4. A graph should appear with concentration on the x-axis and absorbance on the y-axis. BIOL-1406 Lab Manual Page 51 of 118  5. With the cuvette containing the blank (water) in the chamber, click on “Collect” on the top menu. 6. Wait at least 3-5 seconds. A small dot (or triangle) may appear on the screen, but even if you do not see it, after 5 seconds or so, click on “Keep” on the top bar. 7. A menu appears, asking for the concentration of the sample. Type-in 0 (zero, because the water has 0% blue dye) and click OK. 8. Now insert the cuvette with sample #1 of blue dye and wait at least 5 seconds. 9. When the dot appears (or even if it does not), click on “Keep” and enter the concentration you calculated as being present in this sample in the earlier activity on dilutions. 10. Repeat steps 8-10 with samples #2 and #3 of blue dye solutions. 11. Click “Stop” on the top bar menu. 12. Click on the “Linear Fit” icon on the top bar menu. If your sample preparation and calibration were done correctly, you should obtain a straight line. Look for correlation in the box showing the slope. The closer that number is to 1 the better your results. ABSORBANCES: Sample 1: ____________ Sample 2: ____________ Sample 3: ____________ Correlation (R) Value? ________________ QUESTIONS 1. Which SAMPLE had the HIGHEST CONCENTRATION of dye? _____________ 2. Which SAMPLE had the HIGHEST ABSORBANCE READING? ______________ 3. Circle the correct term to describe the relationship between dye concentration and absorbance of light: The higher the concentration of dye, the higher / lower the absorbance. (circle one) References: 1. Eberhard, C., General Biology Lab Manual, 1996, Harcourt 2. Vernier Biology Laboratories (Spectrophotometer Source) 3. Edvotek, The Biotechnology Educational Company (Micropipettors Source) BIOL-1406 Lab Manual Page 52 of 118