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Jce0598 p625 analysis of soft drinks: uv spectrophotometry, liquid chromatography, and capillary electrophoresisIn 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 analy- sis laboratory should accomplish several instructional goals.
First, it must demonstrate the capabilities and limitations ofthe method, as well as the proper procedures for data acqui-sition and computation of results. Students must also be re-minded of the importance of the chemistry of the sampleand how this relates to the analysis. Application of the methodto a commercial product always helps stimulate student in-terest and teaches the extra considerations necessary in a real-world analysis. It is also instructive to analyze the same productby more than one instrumental method.
Analyses of regular and diet soft drinks fulfill all these objectives. The samples are common everyday products, andthey may be analyzed by a variety of means. Soft drink com-ponents have been determined by HPLC with UV detection Figure 1. Structures and UV spectra of 7.83 mg/L caffeine (——), (1–5) for a number of years, and methods utilizing capillary 6.55 mg/L benzoic acid (– – –), and 18.7 mg/L aspartame (- - -) electrophoresis to determine caffeine (6, 7 ) and other com- in 0.01 M HCl. The structures represent the predominant proto-nated forms at pH 2.0. Spectra show little variation with pH.
ponents (8) have been developed. Although certainly not asuseful as LC or CE, multicomponent UV analysis can alsobe used if the product does not contain too many absorbingcomponents.
This paper describes a series of undergraduate experi- ments using these three instrumental methods for the analysisof 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 teach-ing the physical bases and practical applications of the threeinstruments, the experiments stress the chemical nature ofthe sample, especially the acid/base character of the threecompounds and the importance of pH in the design of LC andCE 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, Figure 2. Spectrum of a 1:25 dilution of Mello Yello in 0.01 M HCl.
considering the current interest in “natural” foods.
Multicomponent UV Analysis
Figure 1 shows the structures and spectra of all three compounds in 0.01 M HCl. The absorption profiles of caffeine Multicomponent spectral analysis is described in stan- and benzoic acid are quite different, an indication that this dard analytical texts (9, 10) and is a common experiment in pair is well suited to the method. In some instructional settings the undergraduate curriculum (11). In addition to accessing it may be feasible to use a diet drink and analyze the aspartame the software in the Hewlett-Packard 8450A Spectrophotom- separately by a suitable solid-phase extraction technique.
eter, University of Florida students are required to analyze However, to save time and limit the focus of the data analysis, the data manually using simultaneous equations, as described a nondiet beverage is used. Of the several caffeinated drinks in the references. Because the number of equations must equal that were tested, the best choice was Mello Yello. Colas do or exceed the number of components, manual data reduction not give acceptable results because an appreciable colorant can be unwieldy for a three-component system. To simplify band extends into the UV. The spectrum of Mello Yello is the math, the analysis is limited to two of the compounds, shown in Figure 2. Although there is a small absorption at 300 nm, due probably to colorant, the effect on the resultsis 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- JChemEd.chem.wisc.edu • Vol. 75 No. 5 May 1998 • Journal of Chemical Education In the Laboratory
mental principles of multicomponent analysis, the requirements the aspartame elutes shortly after the caffeine, as shown in of the absorbing system, the choice of wavelengths, and the Figure 3. The most logical explanation is the presence of 45% special features of the HP 8450A diode-array instrument.
methanol, which may increase the fraction of aspartame in The procedure calls for the preparation of a series of caf- the HAsp form (i.e., pKa may decrease). Also, benzoic acid’s feine and benzoic acid standards in 0.01 M HCl, with con- long retention time is indicative of considerable interaction centrations in the ranges 4–20 and 2–10 mg/L, respectively.
of the phenyl group with the stationary phase, and this ef- Students measure the UV spectrum of each standard, a syn- thetic unknown prepared by the instructor, and a 1.000- to The CE experiment is performed on a Hewlett-Packard 25.00-mL dilution of filtered Mello Yello. In consultation 3DCapillary Electrophoresis system, which has an automatic with the teaching assistant, they choose two wavelengths and sample changer and a diode-array detector, using an applied obtain the absorbance data for the simultaneous equations potential of 20 kV with the cathode at the outlet. With this method. To verify that the two spectral profiles are additive, configuration, the migration order is cations first in order of a mixed standard containing known concentrations of both decreasing electrophoretic mobility (µep), neutrals next as a components is also tested, and the spectrum is plotted on group, and anions last in order of increasing µep. To separate the same sheet as the spectral sum of the two contributing the three components, a 0.025 M borate buffer, pH 9.4, is single-component standards. In preparation for the subse- used. At this high pH only Caf is neutral; aspartame and ben- quent LC and CE experiments, students obtain spectra of zoate are both anionic (Asp᎑ and Benz᎑). Because of its larger all components in the buffer systems to be used.
size, Asp᎑ should have a lower µep than Benz᎑. Therefore, stu- While in the laboratory, the students obtain the concen- trations of the two analytes in the synthetic unknown andMello Yello from the HP software. For their laboratory reports,they prepare Beer’s law plots for each component at the twowavelengths and use simultaneous equations to determine theconcentrations manually.
Liquid Chromatography and Capillary Electrophoresis
Initially, the LC and CE analyses were combined into a single experiment. Although this was feasible in terms of labo-ratory time, the average student failed to grasp all the instru-mental and chemical concepts, and the experiment was split.
The three-week soft drink module is scheduled in the orderUV, LC, CE.
In addition to the fundamental concepts of LC and CE, the oral quizzes and written reports stress the chemistry ofthe system, especially the importance of pH in the two sepa- Figure 3. Liquid chromatogram of a caffeine/benzoic acid/aspar- ration processes. The laboratory manual gives students the tame mixture obtained on a 15 cm × 4.5 mm Higgins Analytical literature values for the pKa’s of benzoic acid (4.202 at zero column packed with 5 µm Hiasil C18, using a flow rate of 1.0 ionic strength ) and aspartame (2.96 and 7.37 at 0.15 M mL/min and a mobile phase composition of 45% methanol/55% ionic strength ). The actual dissociation constant for caf- 0.025 M aqueous phosphate, pH 3.0. Other instrument compo- feine is not available in standard references. According to The nents included a TSP P200 pump, a Valco injector with a 20-µL Merck Index, the pH of a 1% solution is 6.9 (14). A 0.01 M loop, a TSP UV 100 detector set to 218 nm, and a SpectraPhysics solution in highly purified water was prepared in this labo- ratory. The readings for the solution and the water were both7 within experimental error. Thus, the students are told thatcaffeine’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 thethree components without exposing the bonded octadecyl(C18) stationary phase to excessive acidity. To understand therelationship of the mobile phase composition to the separation,students must first recognize that components should beneutral to interact with the octadecyl stationary phase andthat compounds are expected to elute in order of decreasingpolarity. As shown above, caffeine (Caf ) is such a weak basethat it is neutral at pH’s higher than about 1. In the pH 3.0 Figure 4. Electropherogram of a caffeine/benzoic acid/aspartame mobile phase, the benzoic acid is also in its neutral protonated mixture obtained on a Hewlett-Packard 3DCE system using a 0.025M borate buffer, pH 9.4. The 33 cm × 50 µm capillary was oper- form (HBenz). On the basis of the pKa data, aspartame exists ated at 20 kV. The detection wavelengths were 272 nm for caf- as an equimolar mixture of the fully protonated HAsp+ and feine, 229 nm for benzoate, and 210 nm for aspartame. Before the zwitterionic HAsp forms. In the oral quiz, students predict laboratory period, the capillary was flushed with 0.10 M NaOH, the elution order to be partially ionic aspartame first, followed water, and the borate buffer. Buffer flushes were included after every by very polar caffeine and less polar benzoic acid. In actuality, Journal of Chemical Education • Vol. 75 No. 5 May 1998 • JChemEd.chem.wisc.edu In the Laboratory
dents expect Caf to migrate fastest, followed by Asp᎑, with Benz᎑ last. As shown in Figure 4, this order is indeed observed.
In addition to explaining the pH effects and predicting the elution/migration orders, students are asked to use the UV spectra to choose wavelengths for the LC and CE analyses during the prelab quiz. For the LC analysis, the chosen wave- length is 218 nm. As Figure 1 shows, although the compo- nents all absorb appreciably at 218 nm, this wavelength doesnot correspond to λ diode-array capability of the CE system allows an optimum wavelength to be used for each component (210 for Asp᎑,272 for Caf, and 229 for Benz᎑).
In the laboratory, students analyze the same solutions on both instruments. First, individual solutions of each com-ponent in water are injected to determine the elution and method (sample pretreatment, injection, peak quantitation).
migration times. On the CE, students also verify the peak This is considered to be a truer representation of the detec- assignments from the spectra obtained by the diode-array tion limit than the value obtained according to IUPAC rec- detector. They next inject a series of mixed standards, each ommendations (17, 18), which utilizes only the fluctuations containing known concentrations of all three components, in the instrument signal. The full EPA protocol is slightly for preparation of the three calibration plots. The standards, modified (see Appendix) to meet the time constraints of the which are prepared by the instructor before the laboratory period, have concentrations in the ranges 40–200 mg/L forcaffeine, 25–150 mg/L for benzoic acid, and 75–600 mg/L 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 con- ther 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 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 ex- To 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 cal- In 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.
They observe that these ratios are close to the corresponding ratios of the absorptivi- ties at the LC wavelength (218 nm) obtained sults for the same samples and results for lent opportunity to evaluate detection lim- its and compare the values for different in- struments. For this exercise, students are limit according to the method specified by the Environmental Protection Agency (15, 16 ) for caffeine, aspartame, and benzoic acid aInstructor’s value for synthetic unknowns; manufacturer’s value for commercial prod- method takes into consideration the statis- ucts, if available. bResults by HP software/simultaneous equations. cAverage of 2 deter- tical 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 In the Laboratory
to comment on how well the Mello Yello system meets the Acknowledgment
requirements for multicomponent analysis, and the extra ab- Purchase of the capillary electrophoresis system was sorption is an obvious point to address. Thus, this analytical facilitated by grant #DUE 9650497 from the National Science 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 1. Presented at the annual meeting of the Southeast Association presence of sodium benzoate (19). However, the additivity test of Analytical Chemists, Athens, GA, October 1996, and the Annual described above produces spectra that match almost perfectly, Meeting of the Florida Sections of the American Chemical Society, and a published UV analysis for pharmaceutical mixtures of caf- feine, sodium benzoate, phenacetin, and Pyramidon showed Literature Cited
good agreement, with relative errors of less than 3% (14 ).
1. Smyly, D. S.; Woodward, B. B.; Conrad, E. C. J.A.O.A.C. 1976,
The LC and CE calibration plots are also very linear, and the results for the synthetic unknown are generally quite 2. Gillyon, E. C. P. Chromatogr. Newslett. 1980, 8, 50–51.
good, with relative errors less than 10% (less than 4%, if 3. Delaney, M. F.; Pasko, K. M.; Mauro, D. M.; Gsell, D. S.; aspartame by LC and caffeine by CE are excluded). For the Korologos, P. C.; Morawski, J.; Krolikowski, L. J.; Warren, F. V., most part, LC and CE analyses of the drinks agree with each Jr. J. Chem. Educ. 1985, 62, 618–620.
other, the major exception being caffeine in Mello Yello, which 4. DiNunzio, J. E. J. Chem. Educ. 1985, 62, 446–447.
produces a significantly higher value by CE. Of the Mello 5. Strohl, A. N. J. Chem. Educ. 1985, 62, 447–448.
6. Mabrouk, P. A.; Marzilli, L. A. Presented at the 212th National
Yello analyses, the LC result is closest to the manufacturer’s ACS Meeting, Orlando, August 1996; Paper No. 276; see CHED value. The high CE result may be due to another neutral co- Newslett. 1996, Fall.
migrating with the caffeine, but the spectral profile shows 7. Conte, E. D.; Barry, E. F.; Rubinstein, H. J. Chem. Educ. 1996,
no obvious deviation from that of pure caffeine.
The LC and CE values for caffeine in Diet Coke may 8. Schuster, R.; Gratzfeld-Hüsgen, A. Hewlett-Packard Publ. #12- also be compared to those reported previously in this Journal: 5963-1122E; Hewlett-Packard: Palo Alto, 1994.
9. Skoog, D. A.; Leary, J. J. Principles of Instrumental Analysis, 4th 92 mg/L (3) and 134 mg/L (5 ) by LC; 124 (7 ) by CE. Agree- ed.; Saunders: Fort Worth, 1992; p 164.
ment with the latter two values is very satisfactory.
10. Harris, D. C. Quantitative Chemical Analysis, 4th ed.; Freeman: 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 11. Williams, K. R.; Cole, S. R.; Boyette, S. E.; Schulman, S. G. J. MDLs by CE are 1.7, 8.5 for caffeine; 0.29, 2.4 for benzoic Chem. Educ. 1990, 67, 535.
acid; and 2.2, 7.5 for aspartame. The LC and CE values are 12. Martell, A. E.; Smith, R. M. Critical Stability Constants; Plenum: quite close to each other, and all six values are within 2 and 13. Martell, A. E.; Smith, R. M. Critical Stability Constants; Plenum: 8.5 on a micromolar basis. The two caffeine MDLs are also remarkably close to the previously reported value of 1.9 mg/L 14. The Merck Index, 11th ed; Budavari, S., Ed.; Merck: Rahway, NJ, 15. U.S. Environmental Protection Agency. In Code of Federal Regu- Conclusions
lations; Part 136, Title 40, Appendix B, Revision 1.11, U.S. Gov-ernment Printing Office: Washington, DC, 1990; pp 537–539.
In the conclusion to the CE report (i.e., after all three 16. Harris, D. C. Quantitative Chemical Analysis, 4th ed.; Freeman: experiments have been performed), students are asked to com- pare the advantages and limitations of the three methods.
17. Winefordner, J. D.; Long, G. L. Anal. Chem. 1983, 55, 712A–
Factors such as the number of analyzable components, MDLs, 18. Skoog, D. A.; Leary, J. J. Principles of Instrumental Analysis, 4th sample size, ease of performance, and accuracy (compared ed.; Saunders: Fort Worth, 1992; pp 7–8.
to manufacturer’s values) are discussed. The most notable 19. Machek, G.; Lorenz, F. Scientia Pharmaceutica 1966, 34, 213–231.
consideration is the number of components, which is clearlya limitation in UV multicomponent analysis. Good students Appendix
note that sample size is another important factor. Although The EPA defines the MDL as “the minimum the MDLs are about equal for LC versus CE, the 20-µL concentration…that can be measured and reported with 99% con- injection loop on the LC can be rinsed and filled with about fidence that the analyte concentration is greater than zero and is 50 µL of filtered drink, whereas the standard CE autosampler determined from an analysis of a sample in a given matrix contain- vials hold about 500 µL. Thus, even though most of the ing the analyte” (15 ). The latter part of the definition indicates sample can be recovered from the autosampler vial, the CE analy- that the MDL must be determined by an actual analysis and is sis requires a total sample volume about ten times that for LC.
strictly valid only for the particular sample conditions. The code The analysis of soft drink components stimulates student also gives the complete protocol, but, for the reader’s convenience, interest and is instructionally useful. The experiments dem- a brief explanation is included here.
onstrate the analytical use of the three instruments, as well First, the analyst must have an estimate of the MDL. The code as their advantages and limitations. The MDL determinations lists several types of estimates, but the two most applicable to these teach an important procedure frequently used in the work- analyses are (i) the concentration giving a signal roughly 2.5 to 5 place but not included in most texts. The experiments also times the baseline noise (method usually used by the students), and reinforce students’ knowledge of acid/base behavior and stress (ii) the lowest concentration in the linear response range (i.e., the the importance of fundamental chemistry in the design of concentration at which the calibration plot shows a noticeable change in slope). Next, a standard containing 1 to 5 times the approxi- Journal of Chemical Education • Vol. 75 No. 5 May 1998 • JChemEd.chem.wisc.edu 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 where s is the standard deviation of the replicate concentration mea- surements and t99 is Student’s t (one-sided) at the 99% confidence The analyte concentration is evaluated from the least-squares level (98% confidence level if a table of two-sided t’s is used) for N response equation (and suitable additional calculations, if applicable).
– 1 degrees of freedom (N = the number of replicates).
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