Laboratory: Ripening Bananas What's Happening and Can We Control It? (This lab was revised by Laura B. Sonnichsen & Sheryl Drake, Parkland College)

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1 Laboratory: Ripening Bananas What's Happening and Can We Control It? (This lab was revised by Laura B. Sonnichsen & Sheryl Drake, Parkland College) Reference: Davis-McGibony, C.M.; Bennett, R.R.; Bossart II, A.D.; Deal, S.T. J. Chem. Educ., 2006, 83, Report Requirement: Answer all of the questions/do all the computations requested in italics. Questions not in italics do NOT need to be answered. You do NOT have to write a formal lab report. You should write your answers into a word processing program and save the file. Go into the Lab Report Ripening Bananas and cut and paste the answers to each question in the appropriate space. (Lab Report Ripening Bananas can be found on the Content page or under Assignments Quizzes.) The data table for Question 1 will be submitted to the Lab Ripening Bananas Question 1 Dropbox (found under Content page or under Assignments -> Dropbox.) Labs not submitted in the appropriate area or not in the appropriate file format will not be graded. Report Scoring: 20 points total. Questions valued as marked. Working with a Partner: You are permitted to work with a partner. If you do so, the experimental data reported may be the same, but you need to write your own answers to each question. At the beginning of each question you should note that you worked with a partner by writing Lab Partner (fill in name). Note: This lab requires drying overnight so please plan ahead accordingly. Goals: 1. Predict which bananas will have the highest sugar content and the highest starch content. 2. Draw valid conclusions on whether the hypotheses were supported or refuted based on gathered data and calculations 3. Use Keto-Diastix reagent test strips and color chart to determine sugar content 4. Apply significant figures and units to measurements and calculations Materials: Bananas need 3 bananas of different ripeness (green, yellow, and brown/black). Plastic cups (6). Screw top test tubes (3) From CHE 106 Lab Kit Funnels from Patriotic Colors kit. Filter Paper from provided supplies (or you may purchase coffee filters) Acetone or Nail Polish remover this is optional (speeds up drying) Distilled or Deionized Water (can be found very inexpensively at grocery store)

2 Pasteur Pipet (2) - From CHE 106 Lab Kit Commercial Glucose Test Strips (2-3) from provided supplies (or you may purchase) Our laboratory uses Bayer Keto-Diastix brand test strips. They are sold 50 strips per bottle. Each test strip contains the following components: 2.2% w/w Glucose Oxidase (Microbial, 1.3 IU); 1.0% w/w Peroxidase (Horseradish 3300 IU): 8.1% w/w Potassium Iodide; 69.8% w/w Buffer; 18.9% w/w Nonreactive Ingredients. The strip is impregnated with the components listed and a blue background dye. The oxygen liberated in the final reaction binds with the dye to produce a series of color changes 30 seconds after wetting the strip with urine (or sugar solution). The test strips must be stored at temperatures between F (15-30 C) and out of direct sunlight. Background: If you are a banana eater, you know that the flavor of a green banana is much different than the flavor of a brown banana. You also know that if you buy bananas well ahead of when you plan to eat them, they may well turn black before you get the chance to eat them. What you may not know is that bananas are shipped to the US as hard, green, sour, unripened fruits. They ship better that way, and they arrive into a distributor's warehouse without bruises. Once they reach the warehouse, the bananas are allowed to ripen by the addition of a small amount of ethylene. Ethylene, a ripening agent, is a simple hydrocarbon gas (H2C=CH2) that ripening fruits make and shed into the atmosphere. Sometimes a wound will cause rapid ethylene production; thus picking a fruit will sometimes signal it to ripen, as will an infection of bacteria or fungi on the fruit. This ethylene signal causes developmental changes that result in fruit ripening. Once the ethylene signal is detected and the banana begins to ripen, new enzymes are made. These include hydrolases to help break down chemicals inside the fruits, amylases to accelerate hydrolysis of starch into sugar (the reason a green banana tastes tart and a ripe banana sweet), pectinases to catalyze degradation of pectin (the glue between cells), and so on. 1 As you may know, you can control the ripening of your bananas (both the skin and the fruit inside) by storing them in various ways. The most common methods of storage are on a banana stand, in the refrigerator, in a cool dark drawer, or in a brown paper bag. Introduction: Each person will choose three bananas to test [green (unripe), yellow (ripe), and black (overripe)] for starch and glucose content. You will discover what happens to the chemical composition of a banana as it ripens. Procedure: For each banana, isolate the soluble sugars and the starch, and then determine the amount of each. In order to do so, you will need to perform Parts 1, 2 & 3 on all bananas (separately, of course). Make sure that your bananas are not in the same state of ripening. 1 For more information on the ethylene signaling pathway the following references have been provided. Bleecker, A.B. and Kende, H. Annual Review of Cell and Development Biology, 2000, 16, Guo, H. and Ecker, J.R. Current Opinion in Plant Biology 2004, 7, Koning, R. E. Plant Physiology Information Website. (accessed October 2005) Sauter, M. Ethylene. (accessed October 2005).

3 Part 1: Isolation of soluble sugars from bananas: 1. Obtain 1 of each: unripe (green), ripe (yellow), and overripe (brown) bananas. The following procedure is written for 1 banana and then you ll repeat it for the other two bananas. However, it is suggested that you perform the procedure on all 3 bananas at the same time instead of one at a time (to save you time.) 2. Weigh out approximately 1 g of banana on a top-loading balance in a small plastic cup (saved from Patriotic Colors Lab). Don t forget to tare the cup before adding the banana. Note: It is not necessary to weigh out exactly 1 g of the banana, however it is important to know the exact mass of your sample. 3. Record the mass of the banana sample on your data sheet. 4. Add 6 ml of distilled water and thoroughly grind the sample using a spoon. 5. Pour the sample from the cup into a screw-top tube. Make sure that you are careful when transferring the banana suspension. You need to get all the liquid & solid into the tube. 6. Rinse the cup with 6 ml of fresh distilled water and add the rinse to the same tube. 7. Tape the tubes inside a top-loader washing machine [Note: the intro lab video shows it being taped to the side but it will likely work better if you tape it to the bottom with the top of the screw-top tube towards the middle], and use the spin cycle to centrifuge the tubes. Keep spinning until all the banana is a solid pellet at the bottom of the tube. Note: If you don t have a washing machine with a vertical tub, you can try taping the tubes to a ceiling fan instead (put the cap end towards the center) and turn on medium. Some students have reported successful results using tubes in a salad spinner or even just tying a string and swinging it over head quickly (be careful!). One set of students creatively even set up their test tubes on cardboard and attached that to a power drill/screwdriver as seen in this picture on the right. 8. Decant (pour carefully) the supernatant (the liquid) into a cup. Make sure that all of the solid stays in the tube (you can use a pipet to hold the solid in). If you can t do this by pouring, use a pipette to transfer the liquid. 9. Wash the pellet (the solid left behind) with 6 ml of fresh distilled water by adding the water to the centrifuge tube or test tube and thoroughly breaking the pellet up with a pipette. Make sure that you don t draw anything up into the pipette. 10. Centrifuge the suspension again in the washing machine (as described above). 11. Add this supernatant to the one from the previous centrifugation (in the same cup) and set the pellet aside to use in Part 2 below. 12. Using a graduated cylinder, carefully measure the volume of the combined supernatants. Record this volume in your data table. Rinse the g.c. before using it for another banana. 13. Pour the solution back into the cup, label it and set it aside for use in Part 3. Part 2: Isolation of the insoluble starch: 1. Obtain 10 ml of distilled water. Add a small portion (approx. 3 ml) to the tube and use the pipette to re-suspend the pellet from Part Obtain a filter paper or coffee filter and prepare it for using as filter paper by spreading it out, then folding in half and then in half again (to give ¼). Cut the outer edge of the filter

4 paper so that it fits in the funnel. You may have a small about above the funnel. Mass the paper and record this mass on the data sheet. 3. Place the filter paper in the funnel and open it to form a cone as pictured to the right. Now filter the re-suspended pellet. 4. Add another small portion of distilled water to the centrifuge tube, rinse, and pour this through the funnel trying to transfer as much solid as possible. Keep rinsing until all of the solid is transferred, or until all of the 10 ml of water is used. 5. If solid remains in the test tube, obtain a second 10 ml of distilled water. Again, using small portions, rinse the tube, and pour this through the funnel until all the solid is transferred or until all 10 ml has been used. 6. Once all of the water has gone through the filter, remove the filter paper from the funnel and unfold it. Lay the filter paper flat on a plate and let dry overnight. Record the mass of the filter paper plus banana solid on your data sheet. DO NOT mass until the filter paper and banana solid are completely dry. (If you wish to speed up the drying process, rinse the sample with acetone after all the water has gone through the filter, but before you remove the filter paper from the funnel.) Part 3: Measuring the amount of glucose in the Banana Extract: Most of the soluble material in your sample is glucose. The riper your banana is the more glucose it should contain. You will be using Keto-Diastix Reagent Test Strips to assay for glucose. 1. Remove one test strip from the packaging. DO NOT touch the test areas of the strip. 2. Dip the test strip into the Banana Extract solution you set aside in Part 1 and remove immediately (drawing the edge of strip against the rim of the beaker or flask). 3. Compare the glucose test area to the Glucose Color Chart (on bottle) exactly 30 seconds after wetting. 4. Record the amount of glucose in the data table. 5. If the amount of glucose exceeds 2000mg/dL, (brown color), then dilute your sample by adding an additional 10 ml of distilled water. Mix the solution well, and retest. Question 1: (12 points) Prepare a table like the one shown below (on the next page) and fill in all of the information. The green text are hints on how to calculate the quantity. Please show your work for the % of glucose and the % of starch in banana extract. Save a copy of this table in.docx,.rtf or.pdf format and submit to the Lab #8 Question 1 Dropbox. See table on next page

5 Banana 1 Banana 2 Banana 3 Banana Type (Green, Ripe, Overripe) Color of Banana Skin Mass of Banana Sample (from part 1, #2) (from part 1, #2) (from part 1, #2) Volume of Banana Extract (in ml) (from part 1, #12) (from part 1, #12) (from part 1, #12) Mass of Filter Paper (from part 2, #2) (from part 2, #2) (from part 2, #2) Mass of Starch and Filter Paper (from part 2, #6) (from part 2, #6) (from part 2, #6) Mass of Starch (= mass of starch and filter paper mass of filter paper) (= mass of starch and filter paper mass of filter paper) (= mass of starch and filter paper mass of filter paper) % Starch in Banana starch/mass of Concentration of Glucose in Banana Extract Total Grams of Glucose in Banana Extract for this step below the table and watch that units cancel correctly! starch/mass of starch/mass of (from part 3, #4) (from part 3, #4) (from part 3, #4) (=volume of banana extract x concentration) % Glucose in Banana glucose/mass of (=volume of banana extract x concentration) glucose/mass of (=volume of banana extract x concentration) glucose/mass of Calculation Hints: To determine total grams of glucose, you will need to do some unit conversions. Before you multiply the volume by the concentration, make sure that the volume is in dl, not ml. Also, record your final answer in grams, not milligrams. for each of these below the table. Question 2: (1 point) Compare your results for % starch for the bananas. What happened to your % of starch as a banana ripens? Is this what should happen? Question 3: (1 point) Compare your results for % glucose for the bananas. What happened to your % of glucose as a banana ripens? Is this what should happen? Question 4: (2 points) List a possible source of experimental error for the % starch results. List a possible source of experimental error for the % glucose results. Be specific for each error. Note Human error is not an acceptable answer.

6 Question 5: (1 point) What type of chemical reaction is taking place (inside the banana) as it ripens? The lab report will ask for a single word answer, you do not need a complete sentence here. Question 6: (1 point) What type of bond is cleaved during this process? The lab report will ask for a single word answer, you do not need a complete sentence here. Question 7: (1 point) Based on your results in this lab, summarize what happens to the chemical composition of a banana as it ripens and how this occurs. Question 8: (1 point) Please give constructive feedback regarding this lab. Were the directions clear? How long did it take you to complete this lab? How does this lab compare to the others - in difficulty, time, enjoyment, & relation to class content? Did it illustrate CHE 106 concepts? What did you gain/learn from doing the experiments that you did not learn from the text alone? What would you keep/recommend in future semesters? What would you change? You should write your answers into a word processing program and save the file. Go into the Lab Report Ripening Bananas and cut and paste the answers to each question in the appropriate space. (Lab Report Ripening Bananas can be found on the Content page or under Assignments Quizzes.) The data table for Question 1 will be submitted to the Lab Ripening Bananas Question 1 Dropbox (found under Content page or under Assignments - > Dropbox.) Labs not submitted in the appropriate area or not in the appropriate file format will not be graded. This lab was revised by Laura Sonnichsen and Sheryl Drake from the lab in the article: Davis- McGibony, C.M.; Bennett, R.R.; Bossart II, A.D.; Deal, S.T. J. Chem. Educ., 2006, 83, You are welcome to copy and distribute the lab so long as credit is given. Ver