In the Laboratory
Analysis of Soft Drinks: UV Spectrophotometry, Liquid Chromatography, and Capillary Electrophoresis1 Valerie L. McDevitt, Alejandra Rodríguez, and Kathryn R. Williams* Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, FL 32611-7200
An experiment for the undergraduate instrumental analysis laboratory should accomplish several instructional goals. First, it must demonstrate the capabilities and limitations of the method, as well as the proper procedures for data acquisition and computation of results. Students must also be reminded of the importance of the chemistry of the sample and how this relates to the analysis. Application of the method to a commercial product always helps stimulate student interest and teaches the extra considerations necessary in a realworld analysis. It is also instructive to analyze the same product by more than one instrumental method. Analyses of regular and diet soft drinks fulfill all these objectives. The samples are common everyday products, and they may be analyzed by a variety of means. Soft drink components have been determined by HPLC with UV detection (1–5) for a number of years, and methods utilizing capillary electrophoresis to determine caffeine (6, 7) and other components (8) have been developed. Although certainly not as useful as LC or CE, multicomponent UV analysis can also be used if the product does not contain too many absorbing components. This paper describes a series of undergraduate experiments using these three instrumental methods for the analysis of components of public interest in commercial soft drinks: caffeine, a central nervous system stimulant; sodium benzoate (determined as benzoic acid), which serves as a preservative; and the artificial sweetener aspartame. In addition to teaching the physical bases and practical applications of the three instruments, the experiments stress the chemical nature of the sample, especially the acid/base character of the three compounds and the importance of pH in the design of LC and CE separations. As part of the data acquisition and analysis, students also determine the method detection limits (MDL) for the three compounds by LC and CE. The concepts of MDL, false positives, and false negatives are especially relevant, considering the current interest in “natural” foods. Multicomponent UV Analysis Multicomponent spectral analysis is described in standard analytical texts (9, 10) and is a common experiment in the undergraduate curriculum (11). In addition to accessing the software in the Hewlett-Packard 8450A Spectrophotometer, University of Florida students are required to analyze the data manually using simultaneous equations, as described in the references. Because the number of equations must equal or exceed the number of components, manual data reduction can be unwieldy for a three-component system. To simplify the math, the analysis is limited to two of the compounds, caffeine and benzoic acid. *Corresponding author.
Figure 1. Structures and UV spectra of 7.83 mg/L caffeine (——), 6.55 mg/L benzoic acid (– – –), and 18.7 mg/L aspartame (- - -) in 0.01 M HCl. The structures represent the predominant protonated forms at pH 2.0. Spectra show little variation with pH.
Figure 2. Spectrum of a 1:25 dilution of Mello Yello in 0.01 M HCl.
Figure 1 shows the structures and spectra of all three compounds in 0.01 M HCl. The absorption profiles of caffeine and benzoic acid are quite different, an indication that this pair is well suited to the method. In some instructional settings it may be feasible to use a diet drink and analyze the aspartame separately by a suitable solid-phase extraction technique. However, to save time and limit the focus of the data analysis, a nondiet beverage is used. Of the several caffeinated drinks that were tested, the best choice was Mello Yello. Colas do not give acceptable results because an appreciable colorant band extends into the UV. The spectrum of Mello Yello is shown in Figure 2. Although there is a small absorption at 300 nm, due probably to colorant, the effect on the results is not detrimental to the overall goals of the experiment. An essential part of the laboratory experience is the group prelab quiz. Questions for this experiment focus on the funda-
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mental principles of multicomponent analysis, the requirements of the absorbing system, the choice of wavelengths, and the special features of the HP 8450A diode-array instrument. The procedure calls for the preparation of a series of caffeine and benzoic acid standards in 0.01 M HCl, with concentrations in the ranges 4–20 and 2–10 mg/L, respectively. Students measure the UV spectrum of each standard, a synthetic unknown prepared by the instructor, and a 1.000- to 25.00-mL dilution of filtered Mello Yello. In consultation with the teaching assistant, they choose two wavelengths and obtain the absorbance data for the simultaneous equations method. To verify that the two spectral profiles are additive, a mixed standard containing known concentrations of both components is also tested, and the spectrum is plotted on the same sheet as the spectral sum of the two contributing single-component standards. In preparation for the subsequent LC and CE experiments, students obtain spectra of all components in the buffer systems to be used. While in the laboratory, the students obtain the concentrations of the two analytes in the synthetic unknown and Mello Yello from the HP software. For their laboratory reports, they prepare Beer’s law plots for each component at the two wavelengths and use simultaneous equations to determine the concentrations manually.
the aspartame elutes shortly after the caffeine, as shown in Figure 3. The most logical explanation is the presence of 45% methanol, which may increase the fraction of aspartame in the HAsp form (i.e., pKa may decrease). Also, benzoic acid’s long retention time is indicative of considerable interaction of the phenyl group with the stationary phase, and this effect may also occur with aspartame. The CE experiment is performed on a Hewlett-Packard 3D Capillary Electrophoresis system, which has an automatic sample changer and a diode-array detector, using an applied potential of 20 kV with the cathode at the outlet. With this configuration, the migration order is cations first in order of decreasing electrophoretic mobility (µep), neutrals next as a group, and anions last in order of increasing µep . To separate the three components, a 0.025 M borate buffer, pH 9.4, is used. At this high pH only Caf is neutral; aspartame and benzoate are both anionic (Asp᎑ and Benz ᎑). Because of its larger size, Asp ᎑ should have a lower µep than Benz᎑. Therefore, stu1
Liquid Chromatography and Capillary Electrophoresis Initially, the LC and CE analyses were combined into a single experiment. Although this was feasible in terms of laboratory time, the average student failed to grasp all the instrumental and chemical concepts, and the experiment was split. The three-week soft drink module is scheduled in the order UV, LC, CE. In addition to the fundamental concepts of LC and CE, the oral quizzes and written reports stress the chemistry of the system, especially the importance of pH in the two separation processes. The laboratory manual gives students the literature values for the pKa’s of benzoic acid (4.202 at zero ionic strength [12]) and aspartame (2.96 and 7.37 at 0.15 M ionic strength [13]). The actual dissociation constant for caffeine is not available in standard references. According to The Merck Index, the pH of a 1% solution is 6.9 (14). A 0.01 M solution in highly purified water was prepared in this laboratory. The readings for the solution and the water were both 7 within experimental error. Thus, the students are told that caffeine’s pKb is ca. 14. An eluent mixture of 45% methanol/55% 0.025 M aqueous phosphate, pH 3.0, gives the best separation of the three components without exposing the bonded octadecyl (C18) stationary phase to excessive acidity. To understand the relationship of the mobile phase composition to the separation, students must first recognize that components should be neutral to interact with the octadecyl stationary phase and that compounds are expected to elute in order of decreasing polarity. As shown above, caffeine (Caf ) is such a weak base that it is neutral at pH’s higher than about 1. In the pH 3.0 mobile phase, the benzoic acid is also in its neutral protonated form (HBenz). On the basis of the pKa data, aspartame exists as an equimolar mixture of the fully protonated HAsp + and zwitterionic HAsp forms. In the oral quiz, students predict the elution order to be partially ionic aspartame first, followed by very polar caffeine and less polar benzoic acid. In actuality, 626
Figure 3. Liquid chromatogram of a caffeine/benzoic acid/aspartame mixture obtained on a 15 cm × 4.5 mm Higgins Analytical column packed with 5 µm Hiasil C18, using a flow rate of 1.0 mL/min and a mobile phase composition of 45% methanol/55% 0.025 M aqueous phosphate, pH 3.0. Other instrument components included a TSP P200 pump, a Valco injector with a 20-µL loop, a TSP UV 100 detector set to 218 nm, and a SpectraPhysics SP4270 integrator.
Figure 4. Electropherogram of a caffeine/benzoic acid/aspartame mixture obtained on a Hewlett-Packard 3D CE system using a 0.025 M borate buffer, pH 9.4. The 33 cm × 50 µm capillary was operated at 20 kV. The detection wavelengths were 272 nm for caffeine, 229 nm for benzoate, and 210 nm for aspartame. Before the laboratory period, the capillary was flushed with 0.10 M NaOH, water, and the borate buffer. Buffer flushes were included after every third sample.
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In the Laboratory
dents expect Caf to migrate fastest, followed by Asp᎑, with Table 1. Spectral Data for Caffeine, Benzoic Acid, Benz᎑ last. As shown in Figure 4, this order is indeed observed. and Aspartame In addition to explaining the pH effects and predicting λ ε a Compound (nm) (103 L/mol cm) the elution/migration orders, students are asked to use the (L/g cm) UV spectra to choose wavelengths for the LC and CE analyses Caffeine 218 51.1 9.93 during the prelab quiz. For the LC analysis, the chosen wave229 25.9 5.02 length is 218 nm. As Figure 1 shows, although the compo272 48.9 9.50 nents all absorb appreciably at 218 nm, this wavelength does Benzoic Acid 218 52.8 6.45 not correspond to λmax for any of the three compounds. The 229 90.1 11.00 diode-array capability of the CE system allows an optimum 272 7.66 0.935 wavelength to be used for each component (210 for Asp ᎑, ᎑ Aspartame 210 29.3 8.61 272 for Caf, and 229 for Benz ). 218 11.09 3.26 In the laboratory, students analyze the same solutions on both instruments. First, individual solutions of each commethod (sample pretreatment, injection, peak quantitation). ponent in water are injected to determine the elution and This is considered to be a truer representation of the detecmigration times. On the CE, students also verify the peak tion limit than the value obtained according to IUPAC recassignments from the spectra obtained by the diode-array ommendations (17, 18), which utilizes only the fluctuations detector. They next inject a series of mixed standards, each in the instrument signal. The full EPA protocol is slightly containing known concentrations of all three components, modified (see Appendix) to meet the time constraints of the for preparation of the three calibration plots. The standards, student laboratory. which are prepared by the instructor before the laboratory period, have concentrations in the ranges 40–200 mg/L for caffeine, 25–150 mg/L for benzoic acid, and 75–600 mg/L Results for aspartame in water. The remaining samples are unknowns: filtered soft drinks, including Mello Yello and several diet The wavelengths chosen for the UV analysis are the λmax’s drinks, a synthetic unknown, and a solution of one packet for the two components: 229 nm (benzoic acid) and 272 nm of Equal (aspartame) in 100 mL of water. As described fur(caffeine). Both compounds obey Beer’s law over the conther below, students determine the method detection limits centration ranges used in the analysis. Table 1 summarizes for the three compounds. To obtain the necessary data, they the absorptivity data, including values at 218 nm and 210 prepare seven replicate dilutions of one of the standards and nm (aspartame only), which are used in the HPLC and CE obtain the peak areas using the same separation conditions experiments. as the drink samples. Typical analytical results are presented in Table 2. Results Data analysis for both experiments starts with preparation for the synthetic unknown show excellent agreement with of six calibration plots of peak area versus concentration for the instructor’s values, although the caffeine result for Mello each component by each method. The least-squares equations Yello is somewhat high. As stated above, this is probably due are used to evaluate the concentrations of the components in to a small absorbance for Mello Yello at 300 nm, where the the unknowns and the aspartame content of a packet of Equal. individual components do not absorb. This undoubtedly exTo emphasize the relationship of the detection systems tends into the UV and accounts for the high caffeine result. to the UV spectrophotometer, students are also asked to calIn the conclusion section of their report, students are expected culate the ratios (Caf:Asp and HBenz:Asp) of the slopes of the LC calibration plots. Table 2. Results of Caffeine/Benzoic Acid/Aspartame Analyses They observe that these ratios are close to Experimental Result (mg/L) the corresponding ratios of the absorptiviValuea Sample Compound (mg/L) ties at the LC wavelength (218 nm) obtained UVb LC CEc from the UV spectra. The CE report inUV Syn Unk Caf 11.12 11.09/11.38 – – cludes comparisons of the LC and CE reBenz 4.15 4.15/4.24 – – sults for the same samples and results for Mello Yello Caf 148 167/176 138 238 Mello Yello by all three methods. Benz – 208/219 181 198 Analysis of the same compounds by LC/CE Syn Unk Caf 101.8 – 103 93 more than one method provides an excelBenz 78.6 – 81 76 lent opportunity to evaluate detection limAsp 156.9 – 172 153 its and compare the values for different in137 133 Diet Coke Caf 127 – struments. For this exercise, students are 149 Benz – – 156 asked to determine the method detection Asp 521 – 504 496 limit according to the method specified by Diet Pepsi Caf 101 – 108 89 the Environmental Protection Agency (15, Benz – – 165 162 16 ) for caffeine, aspartame, and benzoic acid Asp – – 463 475 by both LC and CE. As explained further d Equal Asp 367 – 355 317 in the Appendix to this paper, the EPA aInstructor’s value for synthetic unknowns; manufacturer’s value for commercial prodmethod takes into consideration the statisucts, if available. bResults by HP software/simultaneous equations. cAverage of 2 detertical fluctuations of the entire analytical minations. dSolution prepared by dissolving 1 packet in 100 mL of water. JChemEd.chem.wisc.edu • Vol. 75 No. 5 May 1998 • Journal of Chemical Education
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to comment on how well the Mello Yello system meets the requirements for multicomponent analysis, and the extra absorption is an obvious point to address. Thus, this analytical interference is actually very useful instructionally. There is also some concern about possible interactions between the two components, because the solubility of caffeine in pharmaceutical preparations is known to increase in the presence of sodium benzoate (19). However, the additivity test described above produces spectra that match almost perfectly, and a published UV analysis for pharmaceutical mixtures of caffeine, sodium benzoate, phenacetin, and Pyramidon showed good agreement, with relative errors of less than 3% (14 ). The LC and CE calibration plots are also very linear, and the results for the synthetic unknown are generally quite good, with relative errors less than 10% (less than 4%, if aspartame by LC and caffeine by CE are excluded). For the most part, LC and CE analyses of the drinks agree with each other, the major exception being caffeine in Mello Yello, which produces a significantly higher value by CE. Of the Mello Yello analyses, the LC result is closest to the manufacturer’s value. The high CE result may be due to another neutral comigrating with the caffeine, but the spectral profile shows no obvious deviation from that of pure caffeine. The LC and CE values for caffeine in Diet Coke may also be compared to those reported previously in this Journal: 92 mg/L (3) and 134 mg/L (5) by LC; 124 (7) by CE. Agreement with the latter two values is very satisfactory. The MDLs (mg/L, µM) by LC are 1.2, 6.1 for caffeine; 0.57, 4.7 for benzoic acid; and 1.4, 4.9 for aspartame. The MDLs by CE are 1.7, 8.5 for caffeine; 0.29, 2.4 for benzoic acid; and 2.2, 7.5 for aspartame. The LC and CE values are quite close to each other, and all six values are within 2 and 8.5 on a micromolar basis. The two caffeine MDLs are also remarkably close to the previously reported value of 1.9 mg/L by CE (7 ). Conclusions In the conclusion to the CE report (i.e., after all three experiments have been performed), students are asked to compare the advantages and limitations of the three methods. Factors such as the number of analyzable components, MDLs, sample size, ease of performance, and accuracy (compared to manufacturer’s values) are discussed. The most notable consideration is the number of components, which is clearly a limitation in UV multicomponent analysis. Good students note that sample size is another important factor. Although the MDLs are about equal for LC versus CE, the 20-µ L injection loop on the LC can be rinsed and filled with about 50 µ L of filtered drink, whereas the standard CE autosampler vials hold about 500 µ L. Thus, even though most of the sample can be recovered from the autosampler vial, the CE analysis requires a total sample volume about ten times that for LC. The analysis of soft drink components stimulates student interest and is instructionally useful. The experiments demonstrate the analytical use of the three instruments, as well as their advantages and limitations. The MDL determinations teach an important procedure frequently used in the workplace but not included in most texts. The experiments also reinforce students’ knowledge of acid/base behavior and stress the importance of fundamental chemistry in the design of an analytical method. 628
Acknowledgment Purchase of the capillary electrophoresis system was facilitated by grant #DUE 9650497 from the National Science Foundation. Note 1. Presented at the annual meeting of the Southeast Association of Analytical Chemists, Athens, GA, October 1996, and the Annual Meeting of the Florida Sections of the American Chemical Society, Orlando, FL, May 1997.
Literature Cited 1. Smyly, D. S.; Woodward, B. B.; Conrad, E. C. J.A.O.A.C. 1976, 59, 14–19. 2. Gillyon, E. C. P. Chromatogr. Newslett. 1980, 8, 50–51. 3. Delaney, M. F.; Pasko, K. M.; Mauro, D. M.; Gsell, D. S.; Korologos, P. C.; Morawski, J.; Krolikowski, L. J.; Warren, F. V., Jr. J. Chem. Educ. 1985, 62, 618–620. 4. DiNunzio, J. E. J. Chem. Educ. 1985, 62, 446–447. 5. Strohl, A. N. J. Chem. Educ. 1985, 62, 447–448. 6. Mabrouk, P. A.; Marzilli, L. A. Presented at the 212th National ACS Meeting, Orlando, August 1996; Paper No. 276; see CHED Newslett. 1996, Fall. 7. Conte, E. D.; Barry, E. F.; Rubinstein, H. J. Chem. Educ. 1996, 73, 1169–1170. 8. Schuster, R.; Gratzfeld-Hüsgen, A. Hewlett-Packard Publ. #125963-1122E; Hewlett-Packard: Palo Alto, 1994. 9. Skoog, D. A.; Leary, J. J. Principles of Instrumental Analysis, 4th ed.; Saunders: Fort Worth, 1992; p 164. 10. Harris, D. C. Quantitative Chemical Analysis, 4th ed.; Freeman: New York, 1995; p 526. 11. Williams, K. R.; Cole, S. R.; Boyette, S. E.; Schulman, S. G. J. Chem. Educ. 1990, 67, 535. 12. Martell, A. E.; Smith, R. M. Critical Stability Constants; Plenum: New York, 1974; Vol. 3, p 16. 13. Martell, A. E.; Smith, R. M. Critical Stability Constants; Plenum: New York, 1974; Vol 5, p 111. 14. The Merck Index, 11th ed; Budavari, S., Ed.; Merck: Rahway, NJ, 1989; p 248. 15. U.S. Environmental Protection Agency. In Code of Federal Regulations; Part 136, Title 40, Appendix B, Revision 1.11, U.S. Government Printing Office: Washington, DC, 1990; pp 537–539. 16. Harris, D. C. Quantitative Chemical Analysis, 4th ed.; Freeman: New York, 1995; p 84. 17. Winefordner, J. D.; Long, G. L. Anal. Chem. 1983, 55, 712A– 724A. 18. Skoog, D. A.; Leary, J. J. Principles of Instrumental Analysis, 4th ed.; Saunders: Fort Worth, 1992; pp 7–8. 19. Machek, G.; Lorenz, F. Scientia Pharmaceutica 1966, 34, 213–231.
Appendix The EPA defines the MDL as “the minimum concentration…that can be measured and reported with 99% confidence that the analyte concentration is greater than zero and is determined from an analysis of a sample in a given matrix containing the analyte” (15 ). The latter part of the definition indicates that the MDL must be determined by an actual analysis and is strictly valid only for the particular sample conditions. The code also gives the complete protocol, but, for the reader’s convenience, a brief explanation is included here. First, the analyst must have an estimate of the MDL. The code lists several types of estimates, but the two most applicable to these analyses are (i) the concentration giving a signal roughly 2.5 to 5 times the baseline noise (method usually used by the students), and (ii) the lowest concentration in the linear response range (i.e., the concentration at which the calibration plot shows a noticeable change in slope). Next, a standard containing 1 to 5 times the approxi-
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In the Laboratory mate MDL is prepared, and seven or more aliquots are analyzed by the usual laboratory procedure. Because of the time required to dissolve the solid compounds, the procedure is modified to have students prepare and inject seven replicate dilutions of one of the mixed calibration standards. The analyte concentration is evaluated from the least-squares response equation (and suitable additional calculations, if applicable).
The MDL is given by:
MDL = t99 × s where s is the standard deviation of the replicate concentration measurements and t99 is Student’s t (one-sided) at the 99% confidence level (98% confidence level if a table of two-sided t’s is used) for N – 1 degrees of freedom (N = the number of replicates).
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