Biology 211 Laboratory Sessions #4,5, and 6. Procedures for Spectrophotometers and Enzymes Laboratory

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1 Biology 211 Laboratory Sessions #4,5, and 6 Procedures for Spectrophotometers and Enzymes Laboratory Absorbance Spectrum: Take measurements of absorbance of the blue solution starting at 400 nm, and ending at 740nm. Please write your results in the table on page 5. After you have taken all your readings, please draw the absorption spectrum on the graph paper on that page. Instructions for using the Spectrophotometers to make an Absorbance Spectrum: 1) Allow 15 minutes for the machine to warm up 2) Using the % transmittance knob (the power knob) set transmittance to zero. The cover should be closed and there should be no cuvette in the holder when you do this. You should recheck to make sure that your spec hasn t wandered off of zero about every 5 minutes. 3) Set the correct wavelength. This is the knob on the top of the spec. For the absorption spectrum your first setting should be at 400nm. 4) If your spectrophotometer has a filter that you need to adjust (bottom left side) make sure this is set to the correct wavelength. 5) Insert the blank. This is a cuvette (please see the note below about cuvettes) that includes the same solvent as the sample. (Your dye is dissolved in water). Using the 100% T/ 0A knob, set the absorbance reading to zero. This is called blanking the machine. Blanking subtracts the amount of light that is absorbed or transmitted by the solvent so that you are only measuring the absorbance of the solute. 6) Insert sample. Read and record the results on page 5. For this lab please record results in absorbance. 7) Use the knob on the top of the spec to put in the new wavelength. (For this absorbance spectrum you should take measurements at 40 nm increments 8) Before taking a reading at a new wavelength you must blank the spectrophotometer. Then reinsert the blue dye and record the next reading. 9) Continue changing the wavelength, blanking and recording your reading through 720 nm. Depending on your spectrophotometer you may have to change the filter level when you get to 600 nm. Note about Cuvettes: Do NOT put a test-tube into the spectrophotometer. Always use a cuvette and always place it into the spectrophotometer facing the same direction. Also wipe the outside of the cuvette with a Chemwipe to remove any moisture or fingerprints before placing into the spec. Cleaning the Cuvette: 1. NEVER use a brush to clean the inside of the cuvette. 2. When finished please rinse out your cuvettes with water and place back in the rack 1

2 Concentration Curve: To make a concentration curve you must make dilutions of your solution, measure their absorbance at the wavelength of maximum absorbance and then make a graph with the concentration on the X-axis and absorbance on the Y- Axis. This should produce a straight line. Procedure for making a Concentration Curve: 1) Using distilled water and the blue solution provided; make four different concentrations of the blue solution. 1X (i.e., full strength) 1/2X, 1/4X and 1/8X. Make 10 mls of each solution. 2) Pour approximately 6 mls of each solution into cuvettes. Fill one cuvette with distilled water. This will be the blank 3) Determine the wavelength of maximum absorbance for your blue dye. This should be apparent from your absorbance spectrum. You may want to make several more readings of the solution to determine more accurately the wavelength of maximum absorbance. 4) Set the correct wavelength on the spectrophotometer. If your spec has a filter that needs to be adjusted make sure it is in the correct location. 5) Blank the spectrophotometer. (Instructions are on the previous page if you have forgotten how to blank the machine.) 6) Read and record the absorbance of your various solutions in the appropriate table on page 5. Since you are not changing wavelengths and all the solutions are in the same solvent, you do not need to blank between measurements. 7) Make a concentration curve on graph paper. On the Y-axis record absorbance on the X- axis record the concentrations.. Using a concentration curve to determine the concentration of an unknown substance: 8) Put 6 mls of one of the blue solutions labeled Unknown A, B or C into a cuvette. 9) Set the wavelength on the spectrophotometer to the same as what was used to make the concentration curve. 10) Blank the spec and read the absorbance of your unknown. 11) Mark where the absorbance of this solution falls on the concentration curve. You should be able to determine the concentration of the unknown. 2

3 The Catecholase-Catalyzed Reaction Catecholase Catechol + O 2 Benzoquinone In today s enzyme experiment you will observe the effect of various concentrations of the enzyme catecholase on the production of benzoquinone. The source of the catecholase is potatoes. Blending potatoes in water made an extract that includes the enzyme. Table 1: Reaction Mixtures for catecholast experiment shows the different amounts of enzymes that will be added. You should measure the production of benzoquinone both qualitatively, by observing and recording the color change, and quantitatively, by using the spectrophotometer. To make measurement of absorbance for benzoquinone you should set the wavelength at 540 nm since that is the wavelength that it absorbs maximally. Since the potato extract itself has a color and therefore will absorb some light, we will use a solution of it instead of water as the blank. That way the absorbance measurements will more accurately reflect the production of benzoquinone. This reaction proceeds very quickly, therefore it is important that you follow directions completely and move quickly. Do NOT add the potato extract until you are ready to immediately place the cuvette into the spectrophotometer. You will record the reaction for four minutes. Table 1: Reaction Mixtures For Catecholase Experiment Tube Water (ml) Catechol (ml) Potato Extract (ml) Blank Procedures for The Catecholase-Catalyzed Reaction 1) Turn on the spectrophotometer and follow steps 1-4 on the instructions for making an absorbance spectrum except the wavelength should be set to 540 nm. 2) Make a blank by adding approximately 1 ml of potato extract and 5 mls of water to a cuvette 3) Obtain at least 6 small pieces of parafilm. Use your blank to practice covering and mixing a solution by inverting the cuvette once. You will need to be able to do this quickly. 4) Use a pipette to measure: 3 mls of water into cuvette mls of water into cuvette 2 4 mls into of water cuvette 3 5) Use a different pipette to add 2 mls of catechol into Tubes 1,2, and 3 6) Add 1 ml of potato extract to Cuvette 1, QUICKLY place parafilm over the top, invert once to mix, and record the absorbance. Move fast. DO NOT WAIT FOR THE ABSORBANCE TO STOP CHANGING. This is time 0 for Tube 1. One member of the team should read and record your results in the table provided on page 6 while the second member is doing step 7. 7) Add 0.5 ml of potato extract to Cuvette 2, QUICKLY place parafilm over the top, invert once to mix, and record the absorbance. Move fast. This is time 0 for Tube 2. Record your results. One member of the team should read and record your results while the second member is doing step 8. 3

4 8) Do not add any extract to Cuvette 3, place parafilm over the top, invert once to mix, and measure the absorbance. This is time 0 for Tube 2. Record your results. 9) Every minute for the next four minutes re-blank the machine and take new measurements of the absorbance of all cuvettes. Before placing cuvettes in the spec, invert once to ensure even mixing of the solution. Record results. 10) Graph your results. The graph should have absorbance on the y-axis and minutes on the x- axis. There should be three lines on the graph. Showing your results: Before leaving lab today each group must show me a) their absorbance spectrum. b) their concentration curve and demonstrate how a concentration curve can be used to determine the concentration of an unknown. Each student (not group) should make a graph of the catecholase reaction as described in Step 10 and bring it to lab next Tuesday. Next Laboratory Session: During our next laboratory session we will discuss graphs and writing figure legends. You will answer questions about what you did in this laboratory session and you will plan an experiment that you will perform the following session. 4

5 Results of the absorbance readings for the blue dye Wavelength Absorbance 400 nm 440 nm 480 nm 520 nm 560 nm 600 nm 640 nm 680 nm 720 nm Absorbance Absorbance Spectrum Wavelength (nm) 5

6 Data For Concentration Curve: Concentration Absorbance 1 X 1/2 X 1/4 X 1/8 X Results of Catecholase Experiment: Please record both the absorbance readings and the color at each time. ABSORBANCE / COLOR TIME (minutes) Cuvette 1 Cuvette 2 Cuvette 3 6