Lab 2. ph and Buffers

Transcription

1 Lab 2. ph and Buffers I. Natural ph Indicators The colored pigment in red cabbage (and other red fruits and vegetables) is one of a number of naturally occurring acid-base indicator dyes. The red color of cabbage comes from a molecule called an anthocyanin Figure 1. The anthocyanin group is a large class of water-soluble pigments found in many fruits, vegetables and flowers. These are the pigments which give plants their brilliant colors ranging from pink through scarlet, purple and blue. Many other foods contain anthocyanins including cranberry juice, black currants, strawberries, and the skins of red apples. Some pharmaceutical effects of anthocyanins have been suggested, for example in treatment of cardiovascular diseases and in ophtamology. The antioxidant potentials of anthocyanins are high. One reason that red wine or purple grape juice seem to reduce cardiovascular disease may be from the anthocyanins from the grape skin. The Molecule Figure 1. Structure of a representative anthocyanin Here is the generalized structure for a purplish red anthocyanin molecule. Chemically, anthocyanins are made of a sugar molecule attached to a nonsugar part called anthocyanidin. This anthocyanidin has three rings, two 6-carbon (i.e. benzene) rings linked to another ring containing five carbons and one oxygen. The various anthocyanins differ from one another with respect to (1) the number and type of chemical groups, either hydroxyl groups (OH) or methoxy (OCH3) groups attached to the rings of the anthocyanidin and (2) to the sugar that forms the glycoside linkage (i.e. the bond between the sugar and the anthocyanidin). Anthocyanins are the products of biochemical pathways in plants. That is, there is not a single anthocycanin gene that makes the purple color. Rather, the pigment is produced through a series of biochemical steps, each step dependent on the previous step

2 for its substrate. Since autumn is fast approaching, here is a list of genes needed for the biosynthesis of anthocyanins in Indian corn. The color of Indian corn appears when the FINAL steps of the pathways are complete. The names and abbreviations that appear over the connecting arrows of the biosynthetic pathways are names of the required enzymes needed to complete that portion of the color-producing pathway in Indian Corn. Anthocyanin biosynthesis in Indian Corn.

3 (A colored final product) (A colored final product) (A colored final product) The presence or absence of extra hydrogen ions (H+) in a solution can have a dramatic effect on the molecules present in solution. For example, an enzyme s activity can be altered by loosing or gaining hydrogen ions in its active site. Small ph changes are usually tolerated and in fact can sometimes help certain enzymes work better. However, large ph changes are usually not tolerated and often results in the loss of enzyme activity to do the enzyme s denaturation. Other organic molecules, called ph indicators in the lab, can be altered by gaining or loosing (H+) ions from their structure. Some ph indicators are actually natural products. Litmus paper, actually works from a pigment isolated from a lichen. Pigments typically contain conjugated double bonds (alternating double and single bonds) giving them their spectral properties or colors. Most ph indicators themselves are weak acids, whose state of ionization is based on the ph of the solution. The conjugated bonds in ph indicators are altered by the presence or absence of H+ from their structure, ultimately changing their spectral properties. Like other organic conjugate rings systems, the structure of the molecule changes in its acid or base form. Some flowers such as hydrangea also contain anthocyanins, and this makes their color sensitive to the acidity of the soil in which they grow.

4 In this exercise, you will: 1.) extract anthocyanin, a natural ph indicator, from red cabbage (or another red fruit or vegetable); 2.) test the effect of differing ph solutions on the color of the anthocyanin. PROCEDURE: 1. Take a few leaves of red cabbage and tear or cut them into small pieces. 2. Fill a 250-mL beaker about ¾ full of the cabbage leaf pieces, and add distilled water to the 200 ml mark. 3. Place the beaker in the microwave and carefully microwave for about 3.5 minutes. Be careful not to let the cabbage boil over! 4. Remove the beaker when you have a dark purple extract. This will be used in the next portion of the experiment. 5. In tubes labeled A through G prepare dilutions of HCl, using the following. a. Tube A contains 1 ml of 0.1M HCl (diluted from a 1M stock) What ph is this? b. Tube B contains 1 ml of a 1:10 dilution of Tube A.What ph is this? c. Tube C contains 1 ml of a 1:10 dilution of Tube B What ph is this? d. Tube D contains 1 ml of a 1:10 dilution of Tube C What ph is this? e. Tube E contains 1 ml of a 1:10 dilution of Tube D What ph is this? f. Tube F contains 1 ml of a 1:10 dilution of Tube E What ph is this? g. Tube G contains 1 ml of a 1:10 dilution of Tube F What ph is this? 6. In tubes labeled H through M prepare dilutions of NaOH, using the following: a. Tube M contains 1 ml of 0.1M NaOH (diluted from a 1M stock) What ph is this? b. Tube L contains 1 ml of a 1:10 dilution of Tube M.What ph is this? c. Tube K contains 1 ml of a 1:10 dilution of Tube L What ph is this? d. Tube J contains 1 ml of a 1:10 dilution of Tube K What ph is this? e. Tube I contains 1 ml of a 1:10 dilution of Tube J What ph is this? f. Tube H contains 1 ml of a 1:10 dilution of Tube I What ph is this? THESE ARE YOUR ph STANDARDS! 7. In a microtiter plate (share with your table), transfer 200 ul of each standard into wells marked A-M. 8. To each well add 0.1 ml of cabbage indicator, record/observe the color of each solution.

5 II. Using Indicators to detect ph Changes and determine the ph of Unknown solutions. In this exercise, you will: 1.) Examine how different ph values affect the color of a prepared universal ph indicator and 2.) Use it to determine the ph of unknown samples. PROCEDURE: 1. In the microtiter plate, as before, transfer ph standards again (200 ul each) into wells A-M. 2. Add 200 ul of Unknowns A, and B to separate wells. 3. Add 0.01 ml of the universal indicator solution to each well. 4. Observe the color change. Deduce the ph of the unknowns from their color. Unknown A ph Unknown B ph III. Titration Curves: DO TOGETHER AS A CLASS!! We learned a titration curve can be used to determine the buffering capacity of weak acids and bases. These curves are generated by plotting the ph change of a weak acid or base versus the amount of acid or base added to the solution. Titration curves have a horizontal sigmoidal shape (S-shape) with its point of inflection at the compound s unique pka value. In this exercise, you will generate your own titration data using the Vernier Lab Quest, its ph probe, and two biological compounds. You will be given or prepare a solution then add increasing amounts of NaOH and monitor how the solution s ph values change. The labquest ph probe will automatically record the ph values as you add the NaOH and will also graph the data for you in real time. The program automatically plots ph on the Y axis and ml of NaOH on the X axis. The shapes of the curves should reveal important information about buffering capacity and pka values of the chemicals tested. THE ENTIRE CLASS MUST WAIT FOR INSTRUCTOR TO GUIDE THE SET UP OF THE VERNIER LABQUEST. YOUR PATIENCE IS APPRECIATED! Procedure 1. While you wait for others, prepare for your group 250 ml of 0.1M NaOH from solid NaOH pellets and distilled water. Ware gloves and eye protection. LABEL ALL BEAKERS! 2. While you wait for others, prepare 50 ml of 0.1M HCl by diluting from the 1M stock solution. Ware gloves and eye protection. LABEL ALL BEAKERS!

6 3. Hook up the labquest following the instructor direction. 4. Calibrate the ph probe following the instructor s direction. 5. Obtain a 250 ml beaker and stir bar and place on the stir plate, you will also need 250 ml of distilled water the this part of the experiment. 6. Follow instructor s direction on how to collect titration data for acetic acid and another compound on your group s choosing. IV. Making a Buffer. Use the Send in the Clones poster at the front of the room to answer questions #9 and #10 of the homework. Homework questions: DUE BEFORE THE START OF NEXT WEEK S LAB! The answers to the questions are to be typed using a word processor. A hardcopy of the answers is to be handed in AS WELL AS AN ELECTRONIC COPY OF THE ANSWERS SENT TO MY ADDRESS ( jthompso@ycp.edu) make sure you put an appropriate subject heading on your so that I know it is from you, such as your name and the lab number. QUESTIONS from cabbage extract lab 1. What is the color range of anthocyanin over the different ph values? 2. If you spilled grape juice (or red cabbage juice) on a pink shirt, would you recommend using vinegar (an acid) or soap (a base) to minimize the color? Why? 3. Propose an enzyme from the Indian corn biochemical pathways listed above, whose loss of function could create a non-pigmented ear of corn (like the summertime favorite Silver Queen. Explain your answer. QUESTIONS: from UNKNOWNS

7 4. Using the Universal indicator, describe the colors that developed at each ph value 5. Indicate the ph identity of the unknowns A and B? QUESTIONS from titrating. 6. After titrating with the labquest, present the graph the was drawn for each compound by taking a screen capture in the logger pro software and importing it in to another program (such as power point). Make sure you determine the flattest slope of the tangent values from your titration curve. Record the graph s pka value. How does the ph at the inflection point relate to the pka value for that compound? 7. Do a Google image search of the compounds your group titrated with NaOH. Copy and paste their names and structures here. How do their structures change at low and high ph? How do the reported pka values for your group s chosen molecule and acetic acid compare to your experimentally determined pka values? 8. In your labquest titration plot of acetic acid, what volume of NaOH (in equivalents) is needed to be added so that there are equal amounts of associated and disassociated forms of this acid in solution? 9. Which compound or compounds could work as a buffer if you wanted to make a solution with ph=7.4? List all that apply. Explain your answer.

8 10. Which compound or compounds could work as a buffer if you wanted to make a solution with ph=12? List all that apply. Explain your answer.