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Home , Bromothymol blue, Colorimeter (chemistry), PH indicator

z Getting Back to Basics 0 (& Acidics)

-4 SAM RHODES

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rin introductory college , bases and buffers is given in the lab handout we Downloaded from http://online.ucpress.edu/abt/article-pdf/68/1/24/339926/4451921.pdf by guest on 30 September 2021 classes, most instructors spend some time discussing provide to our students. the importance of - chemistry in normal cellu- 0- lar processes. Concepts such as protein and nucleic Part I. Learning the Basics >4 acid denaturation are common examples used to illus- trate how changes in hydrogen ion concentration may Materials for the Introductory Exercises influence cellular function. In addition, we may note that enzymes and oxygen transportmolecules (such as We set up our labs with six or seven stations; each z hemoglobin) will undergo conformational changes in containing the solutions, glassware, and equipment response to changes in pH. The role of buffers in mod- needed for all exercises. Students work in groups of erating body fluid pH is also considered to be central two or three. Each station will need a pH meter (or to a good discussion of acid-base chemistry. computer-interfacedpH probe). It is important to keep the pH electrode in a dilute pH 7 buffer, and to rinse 0-4 While these topics are essential building blocks the electrode in this solution between each use. We for understanding modern biology, they are often over- purchase commercially-prepared pH packets and looked during exercises. During the past dilute them to one-fifth the recommended strength.

, two years I have developed a series of brief laboratory z . - Stronger buffers may alter the outcome of the experi- exercises that introduce students to the basic concepts ments. If several pH probes are not readily available, of acid-base chemistry and provide practical examples you could modify the exercises by using broad range that apply directly to the study of biology and the pH paper. Each station will also need an aquarium human body. These activities require a pH meter, a few pump with about one meter of tubing and a Pasteur solutions, and simple glassware; and take about 30 pipet taped on the end so that the air from the pump minutes to complete. I have also developed a carbon blows out of the tapered end of the pipet. This appa- dioxide assay that is based on colorimetric changes of ratus will be used to bubble air into solutions in test a pH indicator when exposed to different concentra- tubes. An artificial blood serum (see Recipes below) tions of in stoppered test tubes. The CO, assay is CO, and a of carbonated water (available at grocery easily utilized by undergraduatestudents to investigate stores) are also required at each station. Preparedsolu- differences in concentrations in vari- tions of are available from biologi- ous breath volumes. The assay, while limited in its sen- cal supply houses. The remainder of the materials are provides a visual reminder of the important sitivity, as follows: relationship between blood carbon dioxide partial pressure (PCO,) and pH. Exercises utilizing the CO, * dropping bottle with 0.04% bromothymol blue assay take 45-90 minutes depending on how much * 25 mL original experimentation the students wish to under- * distilled water in squeeze bottle take. The exercises may be used early in the semester to introduce acid-base concepts, or as a supplement to * several Pasteur pipets a respiratoryphysiology section of a course. * Two 50 mL beakers Prior to performing these exercises the students * rack with several test tubes should review acid-base chemistry. An overview of * dilute (0. 1M) (in dropping bottle) * 40% hydrochloric acid SAM RHODES,Ph.D., is Professor-of Biology at Fr-anklin College, Franklin, IN 46131; e-mail: srhodes@franklin * 0.1 M NH4OH in dropping bottle collge.edu. * 0.1 M urea in dropping bottle

24 THEAMERICAN BIOLOGY TEACHER, VOLUME 68, NO.1, JANUARY2006 StudentProcedures for Introductory nia to the first beaker and 10 drops of urea to the second Exercises beaker.Swirl the beakers and measure the pH of each solu- tion again. Record your results on the data sheet. In the following section I have added a note for the The ammonia is a strongerbase and will affect the pH reader (in italics) after each exercise. more than the urea-even thougheach moleculeof urea con- tains twiceas muchnitrogen. This provides a good point of dis- A. The Effect of Carbon Dioxide on the pH of cussionconcerning metabolism and bodyfluidpH. Solutions Obtain two 50 mL beakers. Put about 20 mL of car- E. The Effects of Biological Buffers bonated water in one beaker and 20 mL of distilled water Rinse out two 50 mL beakers. Place 20 mL of distilled in the other. [Note To Students: Carbonated water is just water into one beaker and 20 mL of artificialblood serum water with a high concentration of CO2 dissolved in it.] into another beaker. Now measure and record the pH in Review the use of the pH meter (as described by your each beaker. Next add 5 drops of 0.1 M hydrochloric acid instructor). Now carefully measure the pH of each solu- to each beaker. Swirl the beakers for about one minute to tion. Which solution is more acidic? Which solution is mix the contents. Measureand record the pH of each solu- more basic? Record your results. Use your textbook or tion again. Which solution, pure water or the synthetic Downloaded from http://online.ucpress.edu/abt/article-pdf/68/1/24/339926/4451921.pdf by guest on 30 September 2021 another reference to find the between blood serum, underwent the greatest change in pH when carbon dioxide and water that causes the change in pH. 5 drops of 0.1 M hydrochloric acid were added to each? Record the equation with your results. Save these solu- Which chemical components of the artificialblood serum tions for PartB. are responsible for helping to maintain a relatively con- In our labs, the distilledwater will usually have a pH of stant pH? about 6.8, dependingon how long it has been sitting exposed The bicarbonateand proteinsin the artificialserum func- to atmosphericcarbon dioxide. The carbonated water will have tion as buffers.The pH of the distilledwater will changeto 3 or a pH of about 4. 4 while the pH of the artificialserum will changeto about 6. B. The Use of Color Indicators To Determine pH F. Strong Acids Denature Proteins Obtain a dropping bottle of bromothymol blue color Place 20 mL of the artificialblood serum in a 50 mL indicator. Add 10 drops of the color indicator to each beaker. Carefully add 5 mL of 40% HCI and swirl the beaker and mix by swirling. What color indicates a more beaker to mix the solution. Carefully observe the beaker basic solution? What color indicates a more acidic solu- for the next five minutes. Describe the visible changes that tion? occurred in the artificialblood serum when 40% HC1was The bromothymolblue will turn blue in distilled water added. What do you think happened in the blood serum and yellow in carbonatedwater. to cause this change? The 40% HCIcauses the proteinsto denatureand form a C. Carbon Dioxide Production & Human flocculent,white precipitate.The precipitatetakes at least 20 Metabolism seconds to begin to form. By the end of five minutes, there Add 5 mL of distilled water to two test tubes. Add 10 should be afair amountvisible in the beaker. drops of bromothymol blue to each test tube and mix thor- oughly. Use the Pasteurpipet and aquariumpump to bub- Part 11.Building on the Basics ble air into the first test tube for about one minute. Was The second part of this laboratory session builds on there a distinct color change in the solution? If so, did the the concepts learned in Part1, and applies acid-basechem- solution become more acidic or more basic? Now obtain a istry to a simple assay for carbon dioxide. The carbon diox- Pasteurpipet and use it to blow air from your mouth into ide assay relies on the observation that the color change in the second test tube. Continue blowing for about one bromothymol blue is proportional to the concentration of minute. Was there a distinct color change?Did the solution H+in solution. Quantitativechanges in pH indicator color become more or less acidic? Recordyour results. may be achieved by injecting measured volumes of exhaled Usuallythere will be no changein coloras a resultof bub- breath into tightly stoppered test tubes. We use blood col- bling room air through the bromothymolblue solution. lection tubes ("Vacutainer")with soft injection stoppers However,the student'sbreath will cause the solution to change that are readily available from medical supply houses. The first to greenand then to yellow. tubes are first rinsed with distilled water, tapped dry on paper towels, and then filled with a dilute solution of bro- D. Acids & Bases Come in Different Strengths mothymol blue using an automatic dispenser. Samples of For this experiment a solution of ammonia (0.1 M exhaled air are collected into breath collection bags that ammonium hydroxide) and a solution of urea (0.1 M) were are easily assembled by the students. Small, waste-basket- made up in identical concentrations. Rinse out two 50 mL size plastic bags are attached to cardboard tubes (see beakers and add 20 mL of distilled water to each. Measure Suppliers at the end of the article) with a rubber band. the pH of each solution. Now add 10 drops of the ammo- After filling with exhaled air, the breath collection bags are

BASICS&ACIDICS 25 closed with a "Twistit" rubber down on a piece of paper towel to (see Figure 1 and remove largerdroplets. TwistitStopper Suppliers at the end of the arti- 2. Using the dispenser, fill the cle). These stoppers have holes tubes with 2.5 mL of the bro- that are closed with a thin wall of mothymol blue. The fluid in all rubber that is easily penetratedby Hole tubes should have an identical a needle. Measured volumes of color and volume. If this is not the exhaled air may be withdrawn through stopper the case, empty the tubes, rinse from the breath collection bag them with distilled water, and and injected into the Vacutainer refill them. test tubes. For this task, we use 3 3. Twist the stoppers onto the top mL disposable syringes fitted Nipple with 16 gauge needles. of each test tube so that a snug, air-tightseal is made. Use a felt- tipped marker to label 12 of the Materials for Carbon Figure1. Dioxide Assay tubes (1-12). The"twistit"rubber stoppers may be purchased in 4. You will be injecting precisely * breath collection bag (see 1,2, and 3-hole varieties.The preformed holes pass measured quantities of gas into Downloaded from http://online.ucpress.edu/abt/article-pdf/68/1/24/339926/4451921.pdf by guest on 30 September 2021 text) throughthe stopper but are covered by a thin- each tube. This is accomplished * 16-20 13 X 100 mm wallednipple.These nipples are designed to be using the following procedure: Vacutainers twistedoff, but in our experiments they function Use a syringe to withdraw a pre- * asan extraction port. cise quantity of gas from the * 3 mL syringe with 16 gauge needle * spectrophotometer or set to 565 nm (optional) * automatic dispenser containing dilute bromothymol blue solution (see Recipes) * felt-tipped marker Student Procedures for Carbon Dioxide Assay BreathCollection "Vacutainer"tubes Spectrophotometer A. Collection of Breath Sample Bag withbromothymol orcolorimeter One student should obtain a breath blue collection bag. Remove the rubber stop- per and place it on the counter top. Inhale Figure2. a full breath. Then hold your nose closed TheCarbon Dioxide Assay. Breath is collectedinto while exhaling forcefully and completely a plasticbag and closed with a into the breath collection bag. Try to "twistit"stopper. Measured volumes of gasare transferred to stoppered "vacutainer" empty your lungs completely. As soon as tubescontaining bromothymol blue.Transmittance of tubes may be readwith a spec- you complete your exhalation, close the trophotometerorcolorimeter (565 nm). bag by inserting the special stopper into the cardboard tube. breath collection bag by inserting the needle B. Reacting Standard Volumes of Gas with through the hole in the stopper. The volumes to be Bromothymol Blue used are 0, 0.5, 1, 1.5, 2, and 2.5 mL. Be sure to have two replicates for each volume. In this part of the experiment, carefully measured With the stopper firmly sealed, inject the gas sam- quantities of gas from the breath collection bag will be ple into the test tube. introduced into closed test tubes containing 2.5 mL of dilute bromothymol blue. As the carbon dioxide from your Withdraw the needle from the stopper with one breath mixes with the water in the solution, quick movement. (H,CO3) is formed. The change in pH will cause a propor- Shake the test tube vigorously for 10 seconds. tionate change in the color of the pH indicator. 5. Allow the tube to sit for an additional 10 seconds. 1. Rinse all test tubes with distilled water. Shake out Record its color. See Figure 2 for a summary of the the excess water and then tap the tubes up side procedure.

26 THEAMERICAN BIOLOGY TEACHER, VOLUME 68,NO. 1,JANUARY 2006 6. If you are not going to read the percent trans- mittance (%T) of the tubes with a colorime- Effectof CarbonDioxide on %Tof BromothymolBlue Solutions ter, be sure to leave the stoppers on the 70 tubes. The solutions will retain their colors 65 - for several hours. If you want to make more 60 quantitative measurements, the percent transmittance of the tubes may be read with aP 55- a colorimeter or spectrophotometer (565 = 50- nm). These data may then be plotted to pro- 45- E duce a "standardcurve" of 0/%transmittance E 40 - versus volume of gas injected. Since expired ~35- air contains 4 to 5%/oCO,, the "Volumeof Gas 30 - y=-5.3251x2 + 31.087x + 23.239 Injected" data may be approximately con- 25 R2= 0.9969 verted to CO, values where 0.0 mL = 0.0% - / CO., 1.0 mL = 5% CO,, and 2.0 mL = 10% 20 -,,,, CO., etc. 0 0.5 1 1.5 2 2.5

Typical results for the carbon dioxide assay are VolumeofExhaled Breath Added to a SealedTube (ml) Downloaded from http://online.ucpress.edu/abt/article-pdf/68/1/24/339926/4451921.pdf by guest on 30 September 2021 given in Figure 3. Transmittance, rather than absorbance, was chosen for the Y-axisbecause ol its Figure3. positive slope and more linear relationship. 11equip- Transmittance(rather than absorbance) was chosen for the Y-axis because ment is not available, or time does not permit, the ofits positive slope and more linear relationship tocarbon dioxide concen- students may estimate carbon dioxide concentra- tration.Theline fitted to the curve is quadratic. tions bv comparing the color of experimental tubes to the color of the "standards."So long as they are stoppered, the solutions will maintain their colors Mix in 70 mL of 0.04% bromothymol blue (Ward's for several hours. Catalog Number 38-9108) to the distilled water.Transfer to At this point students may design their own experi- a closed container or an automatic dispenser. ments to investigate questions such as: Artificial Blood Serum Does the concentration of carbon dioxide in Dissolve 9.0 g NaCI,0.2 g CaCI,(anhydrous), and 0.4 exhaled breath increase if one holds one's breath? g KCIin one liter of distilled water. Afterother ingredients Does the air in the trachea(first air exhaled or dead have dissolved, add 0.2 g NaHCO, and 1.0 g glucose. NowA air space) have the same carbon dioxide concentra- add the whites of two fresh eggs (contain a good mixture tion as air in the alveoli (last air exhaled)? of albumins and globulins). Stir with a for Does hyperventilation reduce or increase the con- one hour. For best results, cover and allow to sit at room centration of carbon dioxide in exhaled breath? temperature lor several hours (overnight). The next day Does re-breathingair increase the carbon dioxide you may wish to filteror decant the solution to remove the concentration? undissolved egg proteins. Does the concentration of carbon dioxide change in "Twistit" Rubber Stoppers - Size 5 exhaled air during the course of physical exertion? Available trom NASCO Science (www.enasco.com) For each experiment, the student collects a gas sample Catalog Number SB10820M. into the breath collection bag, and then translers 1 mL of the sample to a test tube containing 2.5 mL bromothymol Disposable Cardboard Mouthpieces for Breath- blue. Be sure to use two replicates for each "unknown" Collection Bags sample. The assay is sensitive to changes of about 1% CO,. Availablefrom Ward's (sold as an accessory for a wet This should be sufficient to answer the questions posed spirometer)-Catalog Number 14 W 5076 (package of above, but the students must work carefullyto obtain con- 100/$ 17.95). sistent results. Accurate measurements of volume, and clean glassware make a big difference. 13 X 100 mm Blood Collection Tubes with Injection Stoppers Vacutainer (Becton Dickinson: no additive, red stop- Suppliers & Recipes per, siliconized-36643 1). Available through Daigger Bromothymol Blue Solution for Carbon Dioxide Discount Lab Supplies (www.daigger.con-i)-Catalog Assay Number MX2047C: package of 100 tubes/$32.77. To remove any excess carbon dioxide from your dis- Adjustable Volume Dispenser tilled water, heat 840 mL of the water almost to boiling. "Repipet Jr." Plastic Dispenser. Available from Place the water in a loosely covered glass bottle to cool. Daigger-Catalog Number MX20512C ($87.26).

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