In the Laboratory
Analysis of Citric Acid in Beverages: Use of an Indicator Displacement Assay Alona P. Umali and Eric V. Anslyn* Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas 78712 *
[email protected] Aaron T. Wright Pacific Northwest National Laboratory, Richland, Washington 99352 Clifford R. Blieden, Carolyne K. Smith, Tian Tian, Jennifer A. Truong, Caitlin E. Crumm, Jorge E. Garcia, Soal Lee, Meredith Mosier, and Chester P. Nguyen College of Natural Sciences, The University of Texas at Austin, Austin, Texas 78712
An indicator displacement assay (IDA) is a technique popularly used in studying molecular recognition. It has been widely used in monitoring host-guest binding events, determining association constants, and in general, the optical detection of various analytes including anions and cations in solution. It is a convenient alternative to traditional chemosensors (Figure 1A), which use hosts covalently attached to an indicator, giving a measurable change in its spectroscopic properties upon interaction with the analyte (1-4). In an IDA (Figure 1B), an indicator is reversibly bound to a host, obviating covalent synthesis of a host-indicator system. Introduction of a molecule capable of forming a similar reversible interaction with the host causes the indicator to be displaced, resulting in a quantitative change in spectroscopic properties of the indicator. The popularity of an IDA over the traditional sensing method has been brought about by the ability of the IDA sensing system to be modified according to its purpose, while not limiting the available indicators that can be used. The assay has been exploited for the recognition of many molecules of biological and environmental importance including citrate, phosphate and pyrophosphate, nucleotides, sugars, amino acids, and peptides (4-18). We describe an undergraduate laboratory experiment using IDA to quantify citric acid in commercially available beverages. Several examples of supramolecular chemistry laboratories and classroom activities have been published in this Journal (19-22); however, the concept of indicator displacement has not been illustrated in an undergraduate chemistry laboratory. The experiment described has been done by first-year undergraduate students1 who were introduced to the concepts of solutions, Beer's law, and UV-vis spectrophotometers prior to performing the experiment.2 The experiment could also be implemented in an upper-level analytical chemistry course. Overview of the Assay An IDA involves initial binding of the indicator to the host, forming a complex in solution. Upon introduction of an analyte, the indicator is displaced from the host via thermodynamically controlled competition of the analyte and the indicator for the host. Displacement of the indicator from the binding cavity of the host results in a change in the detector output. For a colorimetric indicator, the visible spectra can be collected as the analyte 832
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is titrated into the solution of the host-indicator complex. It is important that only the concentration of the analyte changes during titration. This condition is realized by incorporating the host and the indicator, at concentrations that are exactly the same as those in the solution of the host-indicator complex, into the solution of analyte (referred to as the titrant). A change in the wavelength of maximum absorbance is observed, brought about by the change in the protonation state of the colorimetric indicator, as it is released from the host. The displaced indicator is linearly related to the amount of the analyte introduced but reaches saturation as more analyte is added. A binding curve can be obtained by plotting the change in absorbance at the wavelength of maximum absorbance of the free indicator against a parameter such as the concentration of the analyte in the solution. From this binding curve, only the linear portion of this plot is used in this experiment for the quantitative determination of the concentration of the analyte. Unknowns are then determined using this calibration curve. A fresh hostindicator system is used with a known volume of an appropriately prepared unknown, and the change of absorbance at the same wavelength at which the calibration curve was constructed is observed. To illustrate the IDA, citrate contents of various commercially available beverages were determined. The system used for the assay is composed of host 1,3,5-tris(aminomethyl)-2,4,6-triethylbenzene (23), 1, and indicator chromazurol S (24), 2 (Figure 2). The host can form an electrostatic ion-pairing interaction either with citrate, 3, or with chromazurol S at a pH (5.5) where the host is fully protonated and the indicator and citrate are partially deprotonated. The change in the color of 2 arises from the difference in the extent of protonation of the bound species and that of the unbound species in solution. The pKa of 2 is about 5 (24), and hence, the assay mixture was buffered near this pH such that the protonation state of 2 is easily perturbed upon binding. At this pH, citrate is primarily a dianion, not a trianion, and its affinity to 1 is lower than found in a previous study (18). A lower sensitivity of the assay to citrate is therefore expected, but this allows the calibration curve for citrate to be linear over a longer range of citrate concentration. The results from the method described can be compared to that from an acid-base titration method for citrate determination, which could be given as an additional part of the experiment.3
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Vol. 87 No. 8 August 2010 pubs.acs.org/jchemeduc r 2010 American Chemical Society and Division of Chemical Education, Inc. 10.1021/ed900059n Published on Web 06/10/2010
In the Laboratory Table 2. Conditions for the Determination of Unknown Citric Acid Concentration of Beverages components
cuvette solution
titrant
1
1.25 mM
2
0.036 mM
0.036 mM
beveragea
none
12.5% (v/v)
MES, pH 5.5
50 mM
50 mM
methanol
50% (v/v)
50% (v/v)
1.25 mM
a
Citric acid component reconstituted in MES buffer, pH 5.5, 50% methanol.
Figure 1. Traditional chemosensing assay (A) and indicator displacement assay (B).
Figure 2. Neutral structures of 1, 2, and 3. Table 1. Conditions for the Construction of Standard Curve components
cuvette solution
titrant
1
1.25 mM
2
0.036 mM
0.036 mM
3
0 mM
6.00 mM
MES, pH 5.5
50 mM
50 mM
methanol
50% (v/v)
50% (v/v)
1.25 mM
Experimental Section Chromazurol S, citric acid, and MES (2-(N-morpholino)ethanesulfonic acid) buffer can be purchased from SigmaAldrich whereas host 1 is synthesized (23, 25) but can also be purchased from Beacon Sciences. MES-buffered stock solutions of the assay components 1, 2, and 3 are used to prepare two solutions: the cuvette solution, which contains the buffered 1:2 complex, and the titrant, which contains citric acid or the unknown beverage in addition to the 1:2 complex and is placed in a vial. All components of these two solutions are at specific concentrations (Tables 1 and 2). Although septum-capped cuvettes and vials are recommended in the experiment, regular disposable plastic cuvettes and vials equipped with plastic covers can be used to prevent evaporation of methanol. The titrant is gradually added using a microsyringe or a micropipettor to the cuvette solution. Spectra of the cuvette solution from 380 to 750 nm are obtained each time an aliquot of titrant is added. Because the concentrations of 1 and 2 are the same in the cuvette solution and the titrant, only the concentration of citric acid in the cuvette solution changes. The change in absorbance is due
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to the displacement of 2 from the 1:2 complex as citrate binds to the host. The calibration curve is obtained using a titrant containing a citric acid standard (Table 1). The changes in absorbance at 503 nm are obtained and plotted against the corresponding concentration of citric acid in the cuvette solution, giving a binding curve. Only the linear portion of the curve is retained to give the calibration curve. The quantitative determination with beverages is done immediately thereafter. A fresh cuvette solution and titrant containing the unknown beverage are prepared (Table 2). After addition of a known volume of titrant to the cuvette solution, the change in absorbance at 503 nm is obtained. From this change in absorbance, the concentration of citric acid in the cuvette solution is calculated using the linear equation obtained from the standard curve. The calculated value from this equation is used to determine the concentration of citric acid in the original beverage solution. A number of commercially available beverages containing citric acid, such as 7-Up, Mountain Dew, Sierra Mist, and Sunkist, can be used. An aliquot (10 mL) of the beverage is evaporated and the resulting residue is redissolved in MES, pH of 5.5, in 1:1 (v/v) methanol:water to make 10 mL of the solution. Hazards Methanol is extremely flammable and toxic by inhalation. Methanol may be fatal or cause blindness if swallowed. Solutions should be prepared under the hood. Proper laboratory attire must be worn including goggles and gloves. Chromazurol S and 1,3,5-tris(aminomethyl)-2,4,6-triethylbenzene may be harmful to eyes, respiratory system, and skin. Results and Discussion If the instructors prepare the stock solutions before the lab, the assay can be done in one three-hour session.4 The experiment was carried out in groups of two students. If the desired results were not obtained, students were encouraged to repeat the assay and to offer reasons for the failure. Allowing mistakes and giving students time to correct them helped to instill patience, creativity, and confidence in the laboratory. Overall, students were able to obtain the expected and repeatable overlay of spectra during titration using standard citric acid solution (Figure 3). The calibration curve obtained was also repeatable (Figure 4). The overlaid spectra showed how displacement of the indicator was occurring during titration and that 1:1 host:analyte binding occurred as evidenced by the presence of an isosbestic point (1). The displacement was also
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In the Laboratory Table 3. Students' Citric Acid Concentrations of Commercially Available Beverages beverage Pepsi (control)
Figure 3. Student data from the spectrophotometric titration with standard citric acid. The absorbance maximum of the indicator, 2, is shifted when it is displaced from the binding cavity of the host, 1, by citric acid, 3.
av concn of citric acid/mM -0.559 ( 0.150
Sprite
6.33 ( 0.67
Mountain Dew
8.53 ( 1.00
HEB brand lemon-flavored water
12.8 ( 1.5
HEB brand orange-flavored water
11.7 ( 0.5
cuvettes in a succeeding laboratory session. This repetition ensured the ability of first-year students to grasp the concept of IDA. Toward the end of the semester, the majority of the students could independently design IDAs without the need of a “recipe” using other host-indicator systems when examining other analytes. The cuvette and well-plate methods of IDA were used repeatedly in the following semester. Conclusions Indicator displacement assay can be illustrated in an undergraduate chemistry laboratory by using the technique to determine citric acid concentrations in soda pop and other citric acid containing beverages. Students learn concepts such as concentrations, solutions, molecular recognition, and UV-vis spectrophotometry. The experiment served to strengthen these concepts through a real-world application. The experiment could be done in an upper-division undergraduate laboratory but was implemented successfully in a first-year undergraduate laboratory course to address the need to learn the assay as a tool in an authentic research. Acknowledgment
Figure 4. Calibration curve obtained from the spectra in Figure 3.
signaled by the visible change in the color of the solution from yellow to red as titration progressed. This desired result could only be achieved by ensuring that only the concentration of citrate is changing and not those of the host and indicator. Errors were easily attributed to the dilution of the indicator, which occurs if its concentration is not the same in both the titrant and the cuvette solution. To plot the calibration curve, students were taught how to extract absorbance data at 503 nm from the software associated with the spectrophotometer. It is also important to guide the students in removing extraneous points for the calibration curve, which “saturates” as more titrant is added to the cuvette solution. This is done by picking the highest Pearson correlation coefficient that could be obtained from the plot. Confusion regarding the difference between the IDA calibration curve and the Beer's law calibration curve, which was used in a previous lab experiment, was apparent in the students' reports and was therefore clarified in postlab discussions. In determining the concentrations of citric acid by IDA, it was necessary to emphasize that the value obtained using the equation from the calibration curve is not the concentration of citric acid in the beverage but the concentration of citric acid in the cuvette solution.5 Student results are shown in Table 3. The implementation of the experiment described proved effective in introducing IDA in our undergraduate research lab. The experiment was repeated using 96-well plates instead of 834
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The authors acknowledge support from the National Science Foundation and the College of Natural Sciences at the University of Texas at Austin. Participation of students of the Supramolecular Sensors Stream of the Freshman Research Initiative, spring semesters of 2007, 2008, and 2009, is also greatly appreciated. Notes 1. The students were part of the Freshman Research Initiative (http://web3.cns.utexas.edu/fri/), which was instituted to reinvent the undergraduate research paradigm at the College of Natural Sciences, UT Austin. Among the objectives of this NSF-funded program are to broaden undergraduate student access to research and integrate the curriculum with the program. These objectives necessitate the participation of a large number of incoming freshmen and the complete change in the syllabus of courses to address the needs of the students in accomplishing the participating faculty's research objectives. Student participants of the initiative come from about 25% of incoming freshmen at the College of Natural Sciences and are distributed into “research streams”, one of which is the Supramolecular Sensors Stream that started in the spring of 2007, the stream to which the students who performed the experiment described here belong. Research streams include about 30 students and are not a select group of honor students. Students who have participated in the various research streams under the initiative have generally positive research experience, have gone
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on to work in various research groups at UT, and show a great potential for graduate school or careers in research. From three research streams in 2005, the initiative has grown to include 20 research streams that tackle various topics under chemistry, biology, biochemistry, physics, computer sciences, and math. To augment students' background on the topic involved in the experiment, exercises on the underlying principles, such as absorbance spectra, Beer's law, and the preparation of solutions, were provided prior to the experiment. It was hoped to have the students compare the results of the citric acid content obtained from IDA with that from acidbase titration. Because the IDA presented in this article is specific for citrate and acid-base titration will react with any acid present in the sample of beverage, the acid-base titration method is expected to give a higher value of citrate concentration. However, a common mistake during performance of the two techniques was using the prepared beverage sample meant for the IDA in the acid-base titration. For the first-year lab involved, it was eventually decided to separate the acid-base titration experiment into another laboratory session. If the students prepare the stock and sample solutions, additional laboratory time is needed. An explanation on how to calculate the citric acid in the original beverage is included in the student handout provided as supporting information.
Literature Cited 1. Wallace, K. J.; Nguyen, B. T.; Anslyn, E. V. Encycl. Supramol. Chem. 2006, 1, 1–11. 2. Wiskur, S. L.; Ait-Haddou, H.; Lavigne, J. J.; Anslyn, E. V. Acc. Chem. Res. 2001, 34, 963–972. 3. Nguyen, B. T.; Anslyn, E. V. Coord. Chem. Rev. 2005, 250, 3118– 3127. 4. Buryak, A.; Severin, K. Angew. Chem., Int. Ed. 2004, 43, 4771– 4774. 5. Siering, C.; Kerschbaumer, H.; Nieger, M.; Waldvogel, S. R. Org. Lett. 2006, 8, 1471–1474. 6. Folmer-Andersen, J. F.; Lynch, V. M.; Anslyn, E. V. Chem.;Eur. J. 2005, 11, 5319–5326.
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7. Coggins, M. K.; Parker, A. M.; Mangalum, A.; Galdamez, G. A.; Smith, R. C. Eur. J. Org. Chem. 2009, 343–348. 8. Ma, W. M. J.; Morais, M. P. P.; D'Hooge, F.; van den Elsen, J. M. H.; Cox, J. P. L.; James, T. D.; Fossey, J. S. Chem. Commun. 2009, 532–534. 9. Zhong, Z.; Anslyn, E. V. J. Am. Chem. Soc. 2002, 124, 9014–9015. 10. Neelakandan, P. P.; Hariharan, M.; Ramaiah, D. J. Am. Chem. Soc. 2006, 128, 11334–11335. 11. Carolan, J. V.; Butler, S. J.; Jolliffe, K. A. J. Org. Chem. 2009, 74, 2992–2996. 12. Tobey, S. L.; Anslyn, E. V. Org. Lett. 2003, 5, 2029–2031. 13. Lavigne, J. J.; Anslyn, E. V. Angew. Chem., Int. Ed. 1999, 38, 3666– 3669. 14. McCleskey, S. C.; Floriano, P. N.; Wiskur, S. L.; Anslyn, E. V.; McDevitt, J. T. Tetrahedron 2003, 59, 10089–10092. 15. Wiskur, S. L.; Floriano, P. N.; Anslyn, E. V.; McDevitt, J. T. Angew. Chem., Int. Ed. 2003, 42, 2070–2072. 16. Leung, D.; Folmer-Andersen, J. F.; Lynch, V. M.; Anslyn, E. V. J. Am. Chem. Soc. 2008, 130, 12318–12327. 17. Han, M. S.; Kim, D. H. Angew. Chem., Int. Ed. 2002, 41, 3909– 3911. 18. McCleskey, S. C.; Metzger, A.; Simmons, C. S.; Anslyn, E. V. Tetrahedron 2002, 58, 621–628. 19. Gaitano, G.-G. J. Chem. Educ. 2004, 81, 270–274. 20. Hernandez-Benito, J.; Garcia-Santos, M. P.; O'Brien, E.; Calle, E.; Casado, J. J. Chem. Educ. 2004, 81, 540–544. 21. Haldar, B.; Mallick, A.; Chattopadhyay, N. J. Chem. Educ. 2008, 85, 429–432. 22. Crane, N. J.; Mayrhofer, R. C.; Betts, T. A.; Baker, G. A. J. Chem. Educ. 2002, 79, 1261–1263. 23. Wallace, K. J.; Hanes, R.; Anslyn, E. V.; Morey, J.; Kilway, K. V.; Siegel, J. Synthesis 2005, 2080–2083. 24. Ueno, K.; Imamura, T.; Cheng, K. L. Handbook of Organic Analytical Reagents, 2nd ed.; CRC Press: Boca Raton, FL, 1992; pp 53-62. 25. Schmuck, C.; Schwegmann, M. J. Am. Chem. Soc. 2005, 127, 3373–3379.
Supporting Information Available Student handout; notes for the instructor. This material is available via the Internet at http://pubs.acs.org.
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