The ph Ruler: A Java Applet for Developing Interactive Exercises on Acids and Bases

Transcription

1 Q 2011 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Vol. 39, No. 4, pp , 2011 Computer Software The ph Ruler: A Java Applet for Developing Interactive Exercises on Acids and Bases Received for publication, August 23, 2010, and in revised form, January 20, 2011 Isabelle H. Barrette-Ng From the Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4 In introductory biochemistry courses, it is often a struggle to teach the basic concepts of acid-base chemistry in a manner that is relevant to biological systems. To help students gain a more intuitive and visual understanding of abstract acid-base concepts, a simple graphical construct called the ph ruler Java applet was developed. The applet allows students to visualize the abundance of different protonation states of diprotic and triprotic amino acids at different ph values. Using the applet, the student can drag a widget on a slider bar to change the ph and observe in real time changes in the abundance of different ionization states of this amino acid. This tool provides a means for developing more complex inquiry-based, active-learning exercises to teach more advanced topics of biochemistry, such as protein purification, protein structure and enzyme mechanism. Keywords: ph, acid, base, buffers, java, active learning, inquiry-based learning. INTRODUCTION By the time most undergraduate students majoring in biology and chemistry begin an introductory biochemistry course, they have encountered the concepts of acid-base chemistry, ph, and buffering several times. Unfortunately, many of these students still have difficulty applying these concepts to many of the basic problems encountered in biochemistry. Many of the problems encountered by students when they are introduced to acid-base concepts in secondary school have been studied in detail [1 3]. Unfortunately, these problems are mostly perpetuated for more advanced students if fundamental misunderstandings and conceptual weaknesses are not addressed. This is a common experience for many instructors at the postsecondary level, and was documented in detail by a recent study showing that most students in an undergraduate, introductory biological chemistry class had a fragmented knowledge structure of acid-base concepts [4]. These authors found that most students only understood acids, bases, ph, buffers and titrations as abstract concepts. Moreover, students generally lacked the mathematical skills to see how these concepts could be applied to the practical and real-life problems that arise in biochemistry. These difficulties are commonly encountered when teaching the several hundred students enrolled every year in the undergraduate introductory biochemistry courses at the University of Calgary. In addition to these challenges, students from different backgrounds also seem to view acid-base concepts from very different perspectives. For To whom correspondence should be addressed. mibarret@ucalgary.ca. DOI /bmb example, students from kinesiology or physical education are excited to learn how ph changes during exercise affect the binding of oxygen to hemoglobin. However, many of these students lose interest as the dry and abstract details of ph, buffering, titrations and the Henderson-Hasselbach equation are presented in the traditional manner prescribed by most textbooks. In contrast, chemistry majors, who are more familiar with the theory of ph and titrations, struggle to see how changes in ph relate to biochemical and physiological processes. Teaching acid-base concepts to students with such differing backgrounds and interests poses a serious challenge. To address these and other challenges, a wide range of exercises and pedagogical approaches have been described in textbooks and scholarly articles. Current textbooks generally assume familiarity with the acid-base concepts taught in secondary school and introductory chemistry, and present standard definitions of ph and pk a, as well as a description of titration curves to show the meaning of equivalence points and buffering [5 9]. Although the approaches taken in these popular textbooks are effective for some students, many with weaker backgrounds fail to understand these basic concepts. Recently, to provide students with a deeper understanding of acid-base concepts, a number of authors have also developed active-learning exercises aimed at improving the teaching of acid-base concepts [10 13]. Active-learning, inquiry-based exercises have been shown to increase the depth of understanding for many students in the physical sciences, including chemistry and biology [14 16]. However, following a survey of existing approaches for teaching acid-base concepts and considering the difficulties reported by students in my classes, it became appa- This paper is available on line at

2 333 rent that some serious shortcomings still existed. Although many existing exercises help to address some of the deficiencies of more traditional approaches, most rely on mathematical manipulations and calculations that only appeal to a small number of mathematically inclined students. Such an approach is unlikely to help the vast majority of students in introductory biochemistry, because their difficulty with acid-base concepts usually starts from a weakness in mathematics [4]. Unfortunately, there are few exercises designed to make acid-base concepts more intuitive and relatable to real-life experience. To assist with this problem, the simple but novel concept of a ph ruler was conceived and developed to provide a more intuitive framework for understanding ph and acid-base concepts. Then, to extend this idea by providing a quantitative tool for exploring acid-base equilibria, an interactive, graphical Java applet was constructed to facilitate student-centered, self-directed inquiry. Altogether, this new approach provides students with a deeper and more functional understanding of concepts that may otherwise be viewed as an abstract and irrelevant collection of facts to memorize. By presenting these difficult concepts in a more meaningful manner and by engaging students through self-directed, inquirybased exercises, this approach provides students with a more solid foundation for studying more advanced subjects such as ion-exchange chromatography, enzyme catalysis, and ph-dependent physiological processes. METHODS The ph Ruler applet was developed using the Java Standard Edition Development Kit 6 (Sun Microsystems/ Oracle Corporation) [17]. The applet is compatible with a wide variety of Web browsers and computer platforms in which the Java Runtime Environment (JRE) is installed. At this time, the JRE is not a standard part of most operating systems for microcomputers, but it is freely available for download from the Oracle website. The ph Ruler applet is freely available from the following website: The applet uses the Henderson-Hasselbach equation to determine equilibrium concentrations of weak acids and conjugate bases as a function of ph and the pk a values of the weak acids. JSlider components are used to provide a simple graphical interface that allows users to set the values of ph and pk a for titratable groups. The lengths of vertical bar graphs are set in proportion to the relative abundance of weak acid and conjugate base components at different ph values to provide a simple graphical display in real time. K a ¼½H 3 O þ Š½CBŠ=½AŠ (2) ph ¼ pka þ log 10 ð½cbš=½ašþ; where pk a ¼ log 10 ðk a Þ and ph ¼ log 10 ½H 3 O þ Š The Henderson-Hasselbach (HH) Eq. (3) is introduced by nearly all introductory chemistry and biochemistry textbooks, and students are often asked to plug numbers into the HH equation to calculate the concentrations of A and CB for various weak acids at different ph values. This exercise is intended to show students, for example, that [A] ¼ [CB] when ph ¼ pk a. As well, students with a more mathematical and quantitative learning style should be able to see that [A]. [CB] when ph, pk a, whereas [A], [CB] when ph. pk a. Unfortunately, most students taking introductory biochemistry only understand log and antilog calculations at a superficial level and fail to grasp the meaning of these calculations [4]. As a result, most of these students simply try to memorize a few rules and fail to develop an intuitive understanding of the HH equation. To overcome this problem, the concept of a ph ruler was developed. The basic idea is for students to draw out a ruler, with values of 0 and 14 marked at the left and right ends respectively. Then, for any given weak acid, the student marks down the pk a values on the ruler scale (Fig. 1). When asked whether A or CB is the dominant species in solution at a given ph, the student simply marks down the value of ph on the ruler and asks whether the solution is more acidic or more basic than the position marked for the pk a values. Without doing any calculations, the student can use the ruler as a visual cue to help recall that if the ph is to the left of the pk a on the ruler, then the solution is more acidic than the pk a value, and the A form must predominate. In contrast, if (3) RESULTS The ph Ruler Provides a Graphical Aid for Understanding the Henderson Hasselbach Equation If we abbreviate a weak acid as A and its conjugate base as CB, the equilibrium formed between a weak acid and its conjugate base is described by the following set of familiar equations: A þ H 2 O! CB þ H 3 O þ (1) FIG. 1. The ph ruler construct. For a simple monoprotic weak acid (A) and conjugate base (CB) pair, first the position of the pk a is marked on the ruler. Next, the portion of the ruler on the left of the pk a mark is identified as the acidic range of ph values where the A form is most abundant and the portion of the ruler on the right of the pk a mark is identified as the basic range of ph values where the CB form is most abundant. (a) The arrow indicates that at a ph of 3, the solution lies in the ph range where the A form predominates. (b) The arrow indicates that at a ph of 7, the solution lies in the ph range where the CB form predominates.

3 334 BAMBED, Vol. 39, No. 4, pp , 2011 the ph is to the right of the pk a on the ruler, then the solution is more basic than the pk a value, and the CB form must predominate. When the ph ¼ pk a, then both forms must be present in equal amounts. Although this construct may seem simplistic and trivial at first glance, the positive response from many students suggests that the construct is genuinely helpful. The ph ruler concept seems to provide novices with a simple and intuitive picture of how the relative amounts of A and CB can change at specific ph values relative to pk a values. Many students have provided feedback indicating that the ph ruler concept provides a framework to help organize their thinking about the effects of acidity and basicity on the levels of A and CB. Although this is only anecdotal evidence, it is particularly encouraging to see most students quickly drawing ph ruler pictures to help them answer questions during stressful, time-limited exam situations. Presumably, students would spend the time to draw out ph rulers during exams only if the construct was truly helpful. The ph Ruler Helps Visualize the Distribution of Ionization States for Molecules Containing Multiple Titratable Groups Many biologically important compounds, including amino acids, nucleotides, peptides and nucleic acids, contain multiple titratable functional groups (e.g., carboxylate, amino and phosphate groups). Because the biological functions of these molecules are critically dependent on the ionization states of these functional groups, it is important for students to gain an appreciation of how changes in ph affect the net charge and other chemical properties of these molecules. Unfortunately, the complexities introduced by the simultaneous titration of multiple weak acid-base equilibria within the same molecule often leads to confusion. Most students find it particularly challenging to understand the titration of multiprotic biomolecules for the first time. When asked to calculate the distribution of A and CB forms for a multiprotic biomolecule like an amino acid during a titration, many students do not readily grasp the concept that the titration of weak acid functional groups with strong base occurs sequentially in order of increasing pk a values. This situation becomes even more confusing when students encounter the common cases of amino acids like aspartate, glutamate and lysine, where two separate functional groups have similar pk a values. To help explain how the ph ruler construct helps students understand systems with multiple titratable groups, the ionization equilibria of diprotic acids can be described as follows: A þ H 2 O! CB1 þ H 3 O þ CB1 þ H 2 O! CB2 þ H 3 O þ K a;1 ¼½H 3 O þ Š½CB1Š=½AŠ K a;2 ¼½H 3 O þ Š½CB2Š=½CB1Š ph ¼ pk a;1 þ log 10 ð½cb1š=½ašþ ph ¼ pk a;2 þ log 10 ð½cb2š=½cb1šþ In the case of an amino acid like glycine or alanine without a titratable side chain, the fully protonated form is referred to as A and has a net charge of þ1. The zwitterionic form CB1 has two opposite charges on the same molecule and thus has no net charge. The isoelectric point (pi) is the average of the pk a values around the zwitterionic form. In this case, the pi is 2.3 þ 9.7/2 ¼ 6. In this case, the conjugate base form CB2 has a net 21 charge. For the relatively simple case of alanine, the ph Ruler construct helps students visualize the changes in the amounts of the A, CB1 and CB2 forms (Fig. 2). After marking down the values of pk a,1 and pk a,2 for the carboxylate and amino groups on the ph ruler, the student can divide up the ruler into ranges of ph where a different form of alanine predominates. As a result, the student can now clearly see which form is dominant at a specific value of ph. In more complex cases, such as the triprotic amino acids aspartate, glutamate and lysine, or peptides containing titratable side chains, all of the ionization equilibria present can be described in a similar manner, and the ph Ruler becomes an even more powerful tool for keeping track of additional ionization states: A þ H 2 O! CB1 þ H 3 O þ CB1 þ H 2 O! CB2 þ H 3 O þ FIG. 2.The ph ruler for a diprotic amino acid. Marks on the ruler indicate the pk a,1 value for dissociation of a proton from the carboxylic acid group and the pk a,2 value for dissociation of a proton from the amino group. The ranges of ph where the A, CB1 and CB2 forms predominate are indicated. Panels (a), (b) and (c) indicate clearly how the ruler can be used to identify the main form present in solution at ph values of 1, 7 and 11.

4 335 FIG. 3.The ph Ruler Java applet for a diprotic amino acid. Four distinct regions of the applet window are shown. (a) The protonation states of the carboxylate and amino functional groups and (b) the protonation states of a diprotic amino acid are drawn. (c) Immediately below each protonation state, a bar graph is drawn indicating the percentage of the carboxylate group, amino group or form of the diprotic amino acid that is present in solution at a given ph. (d) Java sliders are present to allow the student to set values for ph, pk a,1 and pk a,2. With the pk a,1 slider, students can set the pk a value for the carboxylic acid on the diprotic amino acid. With the pk a,2 slider, students can set the pk a value for the amino group on the diprotic amino acid. As the widget for any of the sliders is dragged by the student, the bar graphs change in length to indicate changes in the abundance of the protonation states of the carboxylate and amino functional groups, as well as the protonation states of the amino acid. CB2 þ H 2 O! CB3 þ H 3 O þ CBðnÞþH 2 O! CBðn þ 1ÞþH 3 O þ The ph Ruler Java Applet Provides an Interactive, Graphical Interface for Developing Active-Learning Exercises One of the main limitations of the original ph ruler concept is that it provides only a qualitative sense of A and CB concentrations. To extend the original concept and provide students with a more quantitative sense of the HH equation, a simple Java applet was developed. One of the strengths of the Java programming language is the abundance of tools for constructing simple and intuitive graphical user interfaces [17]. The sliders and event handlers found in the Java Swing toolkit were particularly useful for constructing a graphical user interface that brings to life the original ph ruler concept. In the simplest embodiment of the applet, students can drag a slider widget along the ph ruler to set the ph relative to the pk a values of the titratable functional groups of a diprotic amino acid (Fig. 3). As the slider is dragged, the ph value is updated, the concentrations of the A and CB forms of each functional group are calculated using the HH equation, and bar graphs are drawn to provide a graphical representation of A and CB concentrations for each functional group (Fig. 4). In addition, the applet calculates and displays the amounts of the A, CB1, and CB2 species present in solution as ph is changed. In a slightly more complex embodiment of the applet, the ionization states of a third titratable group in the side chain are considered in addition to the amino and carboxylate groups (Fig. 5). This version of the applet introduces an additional slider for controlling the pk a value of the titratable group in the side chain and contains additional bar graphs that monitor the amounts of additional ionization states resulting from the presence of the side chain functional group. Even more complex embodiments of the applet could be envisioned to show the effects of additional titratable groups in small peptides or other biologically important molecules. Because the ph values and bar graphs are updated in real time, students can use the applet to see how changes in ph values affect the concentrations of acid and conjugate base forms for each functional group, as well as the concentrations of the different ionization states of the amino acids. By simply dragging the slider back and forth to change the ph of the solution, students can explore for themselves the effects of ph changes on multiple ionization equilibria. Separate sliders can also be used to change the values of pk a for the carboxylate and amino groups to allow students to see how changes in pk a affect distribution of acid and conjugate base forms. The simple and intuitive graphical interface used in the applet encourages students to play with the sliders and see the effects on A and CB forms. Through this

5 336 BAMBED, Vol. 39, No. 4, pp , 2011 FIG. 4. Demonstration of the applet in action. (a) The ph slider (iii) has been set for 1.3. Under these very acidic conditions, 1.0 ph units below the pk a,1 for the carboxylate group, the bar graphs (i) show that 90.9% of the carboxylate group is protonated and 9.1% is deprotonated. The bar graphs also show that 100% of the amino group is protonated, and (ii) 90.9% of the amino acid is fully protonated, whereas 9.1% is zwitterionic. (b) By setting the ph slider (iii) to 3.3, the bar graphs now show that 90.9% of the carboxylate group is now deprotonated and 9.1% is protonated. Again, 100% of the amino group is protonated, and (iii) only 9.1% of the amino acid is fully protonated whereas 90.9% is now zwitterionic. self-driven learning approach, students gradually gain a deeper and more intuitive sense for how acid and conjugate base concentrations change as a function of ph and the pk a values of weak acids. As a result, students see for themselves the physical meaning of the HH equation, rather than memorizing facts that they do not really understand. This experience leads to a deeper level of understanding that can be developed further through more complex exercises. Acid-Base Detective Exercise Utilizing the ph Ruler Java Applet Although the ph Ruler Java applet can be used as a stand-alone tool to provide students with an unstructured and self-guided learning experience, a wide variety of formal exercises can also employ the applet to direct students through more structured investigations of acidbase concepts. For example, the ph Ruler Java applet can be used by students in a pre-lab activity in preparation for an inquiry-based lab exercise on acid-base concepts. In a novel exercise entitled Acid-Base Detective, students are given a sample of an unknown amino acid and asked to investigate its acid-base properties to help determine its identity. This inquiry-based exercise is intended to help students learn more about the properties of amino acids and the differences in structure and acid-base properties of di- and triprotic amino acids. The ph Ruler Java applet is used to supplement the wet-lab exercise by allowing the students to answer questions using model data in the pre-lab (Fig. 6). In the pre-lab portion of the exercise, students are given a data table indicating the net charge of the predominant form of the unknown amino acid over the range of ph values The students are also given a list of three possible amino acids and the pk a values for their side chains. Students are asked to use the applet to set the pk a values for the carboxylate, amino and side chain functional groups of one of the candidate amino acids, and then evaluate the ionization states present in solution as they adjust the ph slider widget to various values. By comparing the distribution of A and CB forms predicted by the ph Ruler Java applet and the data provided for the unknown sample, the student should be able to deduce the identity of the unknown. Although students can identify the unknown through trial and error without a clear understanding of the underlying concepts, the inquiry-based design encourages students to develop a deeper and more intuitive understanding of acid-base concepts through an active-learning approach that relies on their judgment and initiative.

6 337 FIG. 5. Demonstration of the applet for triprotic amino acids. Compared with the applet for diprotic amino acids, the introduction of an additional titratable functional group in the side chain introduces (i) two additional bar graphs showing the distribution of protonated and deprotonated forms of the functional group in the side chain and (ii) three additional bar graphs showing the relative abundance of amino acid species containing different combinations of the protonated and deprotonated states of the side chain functional group in combination with protonated and deprotonated states of the amino and carboxylate groups. With the pk a,1 slider, students can set the pk a value for the carboxylic acid on the triprotic amino acid. With the pk a,2 slider, students can set the pk a value for the side chain functional group on the triprotic amino acid. With the pk a,3 slider, students can set the pk a value for the amino on the triprotic amino acid. (a) The ph slider (iii) has been set for 5.1. Under these mildly acidic conditions, the bar graphs (i) show the percentages of protonated and deprotonated carboxylate group, amino group and side chain. The bar graphs (ii) show the percentages of the various possible ionization states of the triprotic amino acid. (b) The ph slider (iii) has been set for 7.1. FIG. 6. Sample question from the Acid-Base Detective exercise. Students are presented with model data showing the net charge of the main species in solution for an unknown amino acid at ph values Three possible choices for the identity of the unknown amino acid, as well as the pk a values for the titratable groups in each amino acid are also given. Students are asked to identify the unknown amino acid by using the ph Ruler Java applet to calculate the relative abundance of different ionization states for each of the possible choices and to compare the predictions from the applet with the data presented for the unknown amino acid.

7 338 BAMBED, Vol. 39, No. 4, pp , 2011 Informal feedback from students who undertake the computer-based exercise in advance of the wet-lab exercise shows that the use of the Java applet increases confidence and understanding prior to completion of the wetlab. Furthermore, approximately 80% of students are able to correctly identify the unknown amino acid assigned to them in the Acid-Base Detective exercise after using the ph Ruler Java applet. These initial experiences with using the ph Ruler Java applet are highly encouraging and suggest many different ways in which the applet could be applied to enrich the teaching of acid-base concepts. CONCLUSIONS The simple idea of the ph ruler and the embodiment of this idea in the form of the ph Ruler Java applet provide students in introductory biochemistry with useful tools for learning the basics of acid-base chemistry. The idea of the ph ruler provides students with a visual aid to keep track of how changes in ph relative to pk a affect the concentrations of A and CB forms of weak acids. By simply dragging a slider bar along the ph Ruler Java Applet, students can see how concentrations of A and CB forms change as a function of ph. This novel activelearning tool provides students with a more intuitive sense and a deeper understanding of acid-base ionization equilibria that allows them to explore more complex topics in biochemistry with greater confidence. REFERENCES [1] M. Demerouti, M. Kousathana, G. Tsaparlis (2004) Acid-base equilibria, part I. Upper secondary students misconceptions and difficulties, Chem. Educator 9, [2] M. Demerouti, M. Kousathana, G. Tsaparlis (2004) Acid-base equilibria, part II. Effect of developmental level and disembedding ability on students conceptual understanding and problem-solving ability. Chem. Educator 9, [3] M. Drechsler, J. Van Driel (2008) Experienced teachers pedagogical content knowledge of teaching acid-base chemistry. Res. Sci. Educ. 38, [4] D. J. Watters, J. J. Watters (2006) Student understanding of ph. Biochem. Mol. Biol. Educ. 34, [5] D. Voet, J. G. Voet (2005) Biochemistry, 3rd ed., Hoboken, NJ., Wiley, p [6] C. W. Pratt, K. Cornely (2010) Essential Biochemistry, 2nd ed., Hoboken, NJ, Wiley, p [7] D. L. Nelson, M. M. Cox (2009) Lehninger Principles of Biochemistry, 5th ed., New York, W.H. Freeman and Company. [8] R. F. Boyer (2006) Concepts in Biochemistry, 3rd ed., Hoboken, NJ, Wiley. [9] J. M. Berg, J. L. Tymoczko, and L. Stryer (2007) Biochemistry, 6th ed., New York, W.H. Freeman and Co, p [10] R. W. Clark, G. D. White, J. M. Bonicamp, E. D. Watts (1995) From titration data to buffer capacities: A computer experiment for the chemistry lab or lecture. J. Chem. Ed. 72, [11] H. Drossman (2007) Chemical speciation analysis of sports drinks by acid-base titrimetry and ion chromatography: A challenging beverage formulation project. J. Chem. Ed. 84, [12] R. Curtright, R. Emry, R. M. Heaton, J. Markwell (2004) Facilitating student understanding of buffering by an integration of mathematics and chemical concepts. Biochem. Mol. Biol. Educ. 32, [13] E. O. Carvalho, I. L. Nantes (2008) A novel tool to facilitate the learning of buffering mechanism by undergraduate students of the biological area. Biochem. Mol. Biol. Educ. 36, [14] Y. J. Dori, I. Sasson (2008) Chemical understanding and graphing skills in an honor case-based computerized chemistry laboratory environment: The value of bidirectional visual and textual representations. J. Res. Sci. Teach. 45, [15] T. M. Winberg, C. A. R. Berg (2007) Students cognitive focus during a chemistry laboratory exercise: Effects of a computer-simulated prelab. J. Res. Sci. Teach. 44, [16] M. B. Nakhleh (1994) Influence of levels of information as presented by diffferent technologies on students understanding of acid, base and ph concepts. J. Res. Sci. Teach. 31, [17] J. Gosling, B. Joy, G. Steele, G. Bracha (2005) The Java Language Specification, 3rd ed., p. 649.