In the Laboratory
Analysis of Diet Tonic Water Using Capillary Electrophoresis
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An Undergraduate Instrumental Analysis Experiment Harvey B. Herman,* John R. Jezorek, and Zhe Tang Department of Chemistry, University of North Carolina at Greensboro, Greensboro, NC 27402-6170; *
[email protected] Capillary electrophoresis (CE) is useful for a wide variety of analytical problems. The number of recent citations seems to be rising exponentially in the general literature (1). However, only a few papers in this Journal have described experiments for use in undergraduate laboratories (2–6 ). We felt that our students should be introduced to this method and so we developed a new experiment for instrumental analysis, a junior/senior course in our bachelor of science program. In addition to illustrating the principles of the technique, including electrophoretic and electroosmotic flow (EOF), we wanted a procedure that (i) utilized easy-to-acquire samples containing both cations and anions and (ii) would show good separation using an unmodified fused silica column with unsophisticated equipment. The CE experiment would complement our GC and HPLC separation labs done earlier in instrumental analysis.
Figure 1. Typical electropherogram of diet tonic water with phenol as internal standard.
Background A few years ago we purchased a GTI/SpectroVision capillary electrophoresis system with a Linear Model 200 variable-wavelength UV–vis detector. More recently we obtained a Thermoseparations Spectrophoresis Model 100 CE. We already had several integrators in-house. These are very basic systems, two of the least expensive commercial units. We chose an equally simple sample matrix with UV-absorbing compounds, diet tonic water, to illustrate for our students how capillary electrophoresis on a bare fused silica column can separate cations (quinine) and anions (saccharin and benzoate) in a single run. To improve reproducibility, we added a neutral UV-absorbing compound (phenol) as an internal standard. This allowed us to illustrate both the internal-standard concept and electroosmotic flow, since phenol, a neutral species, migrates at the EOF rate. In our system, baseline separation was accomplished in about 12 minutes (Fig. 1). We had the students quantitate one of the analytes (quinine) using a four-point standard addition calibration (Fig. 2). It is possible to determine the other components, but not within the time constraint of this fourhour lab. Note the “extra” peak (saccharin (1) in Fig. 1.) This peak was found to different degrees in all the diet tonic water samples we tested, and even in liquid saccharin sweetener, as well as in saccharin from a laboratory supply house. Its identity is unknown but it affords an opportunity to discuss “impurities” in real samples. Procedure One or more weeks before the laboratory, the students are given a reading list on capillary electrophoresis. The equipment is preassembled and tested by the instructor before the
Figure 2. Four-point standard-addition determination of quinine in diet tonic water using phenol as internal standard. Regression data: (ratio of areas) = 14.2 (added concentration of quinine) + 0.473, r 2 = .997.
lab begins. The capillary (75 µm internal diameter) is about 100 cm long with an optical window 50 cm from the sample end, formed by flame-removing the polymer coating and washing the window with methanol. The instrument is first demonstrated to the students, after which they are free to begin. Volumetric glassware (5-mL flasks) and pipets (2-mL and 1000-µL micropipetter) are supplied along with stock solutions (pH 7, 20 mM/20 mM Na2HPO4/KH2PO4 buffer containing 0.30 g/L phenol; 1.00 g/L quinine sulfate; and commercial tonic water soft drink samples.) For the separation and quantitation of quinine, the students prepare four solutions, each containing tonic water (2.00 mL) and phosphate buffer
JChemEd.chem.wisc.edu • Vol. 77 No. 6 June 2000 • Journal of Chemical Education
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In the Laboratory
containing phenol (2.00 mL). In the last three, additional amounts of quinine are added (50, 100, and 150 µL) so the standard addition method can be used to find the concentration of quinine in the diet tonic water sample. Deionized water is used throughout for solution preparation and dilution to the mark in the 5 mL flask. Before use, all solutions are filtered through 0.45-µm membrane filters. The capillary and end vials are filled with the so-called run buffer (20 mM phosphate without phenol). After current equilibration, one end of the capillary is put into the sample and a 5–10-second electrokinetic injection is used to introduce the sample into the capillary. The capillary is then returned to the run buffer vial. A setting of 19 kV is used for both injection and run. In the pre-lab lecture, we discuss the possibility of component discrimination when using electrokinetic injection and other methods of sample introduction. After the electropherograms and areas are obtained using the integrator, the quinine/phenol area ratios are calculated manually. The students then check their data using a plotting program. They have time to repeat a sample analysis before leaving the laboratory if one of the points seems off the line. Although students work in groups of three or four on this experiment and work on their calculations together, the reports are done individually. Outside of class, students run a linear regression program, typically with a spreadsheet, and make annotated plots of their data. They use the slope and y intercept from the regression to calculate the negative “x” intercept, whose absolute magnitude is the concentration of quinine sulfate in the diluted sample. In our case, we are using 5-mL volumetric flasks and 2-mL tonic samples, so the intercept result is multiplied by 2.5 to calculate the value in the undiluted sample. For example, in Figure 2 the x intercept was ᎑0.033 g/L. Multiplying the absolute magnitude by 2.5 gives a value of 0.083 g/L quinine sulfate in the original tonic water solution. In the pre-lab, students are intentionally not reminded to use a dilution correction because this concept was covered in several earlier experiments. The FDA standard (7) for carbonated beverages such as tonic water is “Not to exceed 83 parts per million, as quinine.” Since we are using quinine sulfate as our standard, which is not 100% quinine, to calculate the actual amount of quinine for comparison with the FDA standard, multiplication by the ratio of molecular weights (648/783, or 0.83) is required.
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Typically, we measure somewhat less than the maximum allowable, in this case 0.069 g/L or 69 ppm. The students also were asked to calculate theoretical plate values for quinine and compare the efficiency of CE (104/m) to HPLC (5 × 103/m). They can use their HPLC value from an experiment done earlier in the semester. Conclusion We have used this experiment for four years now and it has worked well every time. It illustrates several important concepts: the use of an internal standard; electroosmotic and electrophoretic flow; standard addition; dilution corrections; proper report preparation; and efficiency comparison to HPLC. We feel it is a worthwhile addition to our analytical curriculum. Acknowledgment We wish to thank Keqin He for preparing Figure 1 for publication. W
Supplemental Material
Supplemental material for this article is available in this issue of JCE Online. The student handout includes a complete description of the experiment. Literature Cited 1. Beale, S. C. Anal. Chem. 1998, 70, 279–300. 2. McDevitt, V. L.; Rodriguez, A.; Williams, K. R. J. Chem. Educ. 1998, 75, 625–629. 3. Thompson, L.; Veening, H.; Strein, T. G. J. Chem. Educ. 1997, 74, 1117–1121. 4. Contradi, S.; Vogt, C.; Rohde, E. J. Chem. Educ. 1997, 74, 1122–1125. 5. Vogt, C.; Contradi, S.; Rohde, E. J. Chem. Educ. 1997, 74, 1126–1130. 6. Conte, E. D.; Barry, E. F.; Rubinstein, H. J. Chem. Educ. 1996, 73, 1169–1170. 7. Quinine. Code of Federal Regulations, Part 172, Section 575, Title 21, 1998; pp 60–61.
Journal of Chemical Education • Vol. 77 No. 6 June 2000 • JChemEd.chem.wisc.edu
