Preparing Solutions in the Biochemistry Lab An Essential, Basic Skill John M. Risley The University of North Carolina at Charlotte, Charlotte, NC 28223 One of the most important, albeit least glamorous, aspects of laboratory research is making solutions. This is particularly cmciil in biochemical expiriments where pH, ionic strength, substrate concentration, etc. can be significant factors. No matter how well-desiened a n ex~eriment might be, if solutions are improperly prepared,then the ex~eriment mav fail. I t mav be uninter~retable. I t mavlead to incorrect interpretation?, or it may not bc reproducihle. In lnborntorv courses thrrc is usuallv onlr sufficient time to demonstrate a limited number of concepts. Due to these time constraints, solutions are prepared for use by students. Unfortunately, this can short-change a student. I t is my experience that when upper division undergraduate students and graduate students first begin research, few have acquired the skill of preparing solutions for biochemical experiments. Yet, this is a n essential, basic skill. In a suwey of private and public biotechnology companies, the number one basic skill expected of new employees in research positions is the ability to prepare solutions, buffers, media, etc. (1).In order for our biochemistry students to gain experience in the preparation of biochemical solutions. I wrote a n introductorv laboratorv exercise. which is described below. Chemistry students h i d some experience with solution preparation in quantitative analysis, but, in general, biology students had no experience with solution d re oar at ion. Both mouDs of students benefitted from the iaboratory exercise: The results were reinforced throughout the semester as students routinelv and successfullv prepared solutions for use in other laboratory exercises that did not require extensive laboratory time.
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Experimental Each student prepared the following solutions. 1. 200 mL each of 0.25 M HCI and 0.25 M KOH 2. 100 mL of l-butanol:formic acid:water(100:30:25j 3. 100 mL each of primary standards for calibration of pH meters a. pH (25 "C)= 4.01; 0.05 M potassium hydrogen phthalate b. pH (25 C)= 6.86; 0.025 M potassium dihydrogen phosphatel0.025M disodium hydrogen phosphate c. pH (25 'C) = 9.18: 0.01 M sodium tetraborate 4. 100 mL of phosphate-buffered saline: 10 mM phosphate buffer, pH 8.0 containing 0.9%(wlv)NaCl 5. 100 mL of 10%(wlv)sucrose
Caution: Handle reagents and solutions of HCl, KOH, and formic acid with care. Follow all safety precautions and procedures. These solutions are prepared a s described below. 1. a. 0.25 M HCI: 4.31 mL of commercial HC1 reagent (concen-
trated HCIj in water to give 200 mL of solution b. 0.25 M KOH: 2.81 g of solid KOH dissolved in water to
give 200 mL of solution of 1-butanol, 19.4 mL of formic acid, and 16.1mL of water are mixed 3. a. 0.05 M potassium hydrogen phthalate: 1.021 g of KHP dissolved in water t o give 100 mL of solution
2. 64.5 mL
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Journal of Chemical Education
b. 0.025 M potassium dihydrogen phosphatel0.025 M disodium hydrogen phosphate: 0.340 g of KH2P04and 0.355 g of Na2HP04dissolved in water tc give 100 mL of solution c. 0.01 M sudlum tctrabornte. 0 381 :1 of Na2H,07 ' 10II20 d~srolvedin water ro e v e 100 mL ofsolutmn 4. Phosphate-buffered saline: 0.009 g of NaH2P04' H20 and 0.132 g of Na2HP04dissolved in water ta give 100 mL of solution. The pH is checked to be 8.0. Solid NaCl(O.9 gj is dissolved. 5. 10%(wlv) sucrose: 10 g of sucrose dissolved in appmx. 92 mL of water to give 100 mL of solution. Discussion Some manuals for biochemical laboratory courses contain brief discussions on solutions and their preparation (2-6)in the context ofgeneral techniques, incorporated into experiments, or in an appendix. Often the presentation is in the form of a "handbook" where standard recipes are given, or various solutions are prepared by mixing standard solutions a s listedin tables. These arevery convenient ways to prepare solutions and are routinely used in research labs. However, a basic assum~tionwhen using these 'renpcs" is that the indiwdual understands the fundamental basis for the pre~arationofsolutions and should beable to modify a n e4sti;lg "recipe" or to prepare a completely new solution a s experiments dictate. Stenesh (6)in E x ~ e r iment 1outlines the preparation of two standard solutions from reagents for the preparation of a buffer. Alexander e t al. (7) present a n experiment for students to prepare buffers from reagents; students are required to calculate qnantities of reagents needed for the preparation of different buffers. Otherwise, this subject apparently is not formally presented to students as laboratory exercises. The f r s t experiment in our biochemistry lab was the preparation h i each student of the right sol;tions given in the Experimental section above. Students were shown the reage& bottles for each reagent and then had to calculate the quantity of reagent required to prepare each solution. The proper storage of each solution was also discussed. The conversion of the percentage HC1 given on the reagent bottle of concentrated HC1 into a useful quantity the student could use to prepare 200 mL of 0.25 M HCI was quite a challenge for the students; it required information not seen or used by the students since general chemistry two or more years earlier. There was insufficient laboratory time to standardize the solutions of HC1 and KOH for use in a subsequent experiment, so standardization was briefly discussed; the combined class solutions of HC1 and KOH were standardized by our preparation lab, and the values were within 10% of 0.25 M for each. The problem first encountered by students in the preparation of l-butano1:formic acikwater (100:30:25) was how to prepare 100 mL of solution when the numbers in parentheses did not add up to 100 mL. Once the "ratio" problem was understood. students emressed amazement a t its simplicity. The preparation of the primary standards for calibration of pH meters was graded on how closely the student came
to the values for NBS standards used to calibrate a pH meter. Students' results were routinely within 0.02 pH units of the NBS standards. Students with results outside this range were required to prepare solutions again. These solutions were used by the students during the semester to calibrate pH meters whenever it was required in other experiments. The first question of students preparing these solutions was whether or not waters of hydration had to be included inmolar mass calculations for hydrated salts. The second question was how a solution wuld be 0.025 M potassium dihydrogen phosphate and 0.025 M disodium hydrogen phosphate. The first impression and impulse of these students was to prepare two separate solutions of 0.025 M each, thinking that either one would give pH 6.86; the second impression was to combine the two separate solutions of 0.025 M each to give one solution. That one solution cannot have more than one solute is a common misconception of students. Apparentlystudents are so used to seeing a solution of one reagent that the concept of more than one reagent in a solution is at first quite disturbing. The students were asked to prepare phosphate-buffered saline (PBS) in two steps. First, they prepared 10 mM phosphate buffer, pH 8.0. The preparation of this buffer required a decision by each student on which phosphate salt(s) to use, which for many was not trivial. The use of a pH meter was reviewed at this time. Second, solid NaCl was added to 0.9% (wlv) to this buffer to make PBS. Students were told to check the pH of the buffer to see what effect ionic strength has on the pH of a solution; students were quite surprised by the result. Students were required to calculate the ionic strength of the phosphate buffer and of the PBS solution. The first solution of 10%(wlv) sucrose prepared by many students was made by dissolving 10 g of sucrose in 100 mL of water. To their surprise, these students had more than 100 mL of solution. The students were asked to rewrd the volume of water required to prepare a 10% (wlv) sucrose
solution. This was an extremely valuable lesson to these students. Although this expetiment is rather simple, it illustrates an essential, basic skill needed in any research laboratory The procedure for making solutions was reinforced during the semesterwhen students were requ~redto prepare tho& solut~onsneeded for other experimentx that &d not requlre extensive laboratory time. The terminology used 6 describe various biochemical solutions was discussed as reauired in the subseouent laboratorv exoeriments. Students were continually rehnded to observe all safety procedures when handling and solutions. The students een- reagents erally were cautious and conscientious when preparing these solutions because they understood that the success of their experiments would be based in part on the solutions they were preparing, and, as a result, few problems were encbunter'd. This experiment was also used to refresh students on the use of pipets, the analytical balance, volumetric glassware, and-the pH meter.-student comments were quite favorable, and at the end of the semester students had acquired this skill and appreciated the efforts needed to prepare solutions. Thus, although the topic of solutions is discussed extensively in classroom lectures, it is still very important for students to have experience in the laboratory preparation of solutions.
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Literature Cited 1. Allen, W. E.; Meacher, P.Biofechnology 1888,6,1029-1032. 2. Beeker, J. M.: CaldweU, G. A,; Zsehgo, E. A. Blofechno1ogy:A Laborofow Course: AcademiePress: Ssn Diem. .. CA. 1990: Exercise 5.. Aooendices 3.10.17. . . 3. Rob*. J. F.; m i t e , B. J. Biochemiml T~chniquos:Theory und Pmcflcp; Waveland Ress: RosoedHeizhts. IL. 1990: Chaoter 2. 4. Mineh, M. J.Erpcrimnts in Blochemistry: Pmjecfs ?d P l o d u m s ; RenticeHall: Englewood Cliffs, NJ, 1989; Chapter 2. 5. Plummer, D. T. An Inlraduction lo Pmctical Biochemi~lry,3rd ed.: Mffimw-Hill: Landon, 1981; Chapter 2. 6. Stenesh, J. Ezp~rimnfalBiochemistry;A U p and Bacm: Boston, 1984: Section I, Experiment 1, AppndkB. 7.Alemdar, R.R.;Gfllth8.J. M.:Wi1kinson.M. L.BoaleBlocksmleol Mdhals: Wiley: New York, 1985; Chapter 2.
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Volume 68 Number 12 December 1991
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