In the Laboratory
Determination of Caffeine in Beverages by Capillary Zone Electrophoresis An Experiment for the Undergraduate Analytical Laboratory Eric D. Conte,1 Eugene F. Barry,* and Harry Rubinstein Department of Chemistry, University of Massachusetts Lowell, Lowell, MA 01854 Since the benchmark paper of Jorgenson and Lukacs outlining theoretical and experimental approaches to high resolution electrophoresis in small i.d. glass capillaries (1), capillary electrophoresis (CE) has enjoyed spectacular growth. The number of publications on CE increased from 90 in 1983 to over 300 in 1991 (2). In this time period, distinct modes of CE became defined, including capillary zone electrophoresis (CZE), capillary gel electrophoresis, micellar electrokinetic chromatography, capillary isoelectric focusing, and capillary isotachophoresis (2–4). With the arrival and acceptance of a powerful analytical technique there is an inherent time lag between the introduction of research instrumentation and commercial availability of its more affordable educational counterpart. We describe below the details of construction of a CE apparatus suitable for instructional purposes and a companion undergraduate analytical laboratory experiment, the determination of caffeine in soft drink beverages using an internal standard. Analyte zones are monitored with a UV absorbance detector. The application of an analytical technique to real samples serves to enhance student interest and maintains relevancy and vitality in the program. Certain individuals may be sensitive to specific compounds in consumer products. It is important to quantify these analytes in food products in order to monitor their intake. Caffeine is one such compound. It has been shown to produce abnormal behavioral effects such as hyperactivity and depression (5). The drug may also be linked to myocardial infarction and arrhythmia (6). On the positive side, caffeine may protect against viral induction (7) and chemical carcinogenesis (8). Determination of caffeine in beverages by spectrophotometric procedures requires an extraction procedure, which can prove time-consuming. Although the corresponding determination by HPLC allows for a direct injection (9– 11), CE provides several advantages such as extremely low solvent consumption, smaller sample volume requirements, and improved sensitivity. Experimental Procedure
is opened. Ten centimeters from the cathodic end of the capillary, the protective polyimide coating on the capillary is removed by the flame of a butane lighter, and this stripped segment is inserted into the flow cell compartment of a UV HPLC detector (Waters Model 440, Milford, MA). The HPLC detector modified in this fashion permits on-line CE detection in either the absorbance or millivolt display mode. A Zn light source with the appropriate filter permits the 214 nm line of Zn to serve as the analytical wavelength. All sample solutions are injected by gravity for 5 s at the anodic buffer location, which is positioned approximately 30 cm above the cathodic buffer reservoir for the injection. An internal standard is added to sample solutions to overcome the error associated with a variation in injection volume. A diagram of the apparatus is shown in Figure 1. Electropherograms are collected with an IBM-compatible 286 PC equipped with LabCalc data acquisition software (Galactic Industries, Salem, NH). Alternatively, an Hg source (254 nm) or a Cd lamp (229 nm) instead of a Zn lamp may be used in the detector.
Reagents and Materials Caffeine and sodium borate were purchased from Mallinckrodt, phosphoric acid from Fisher Scientific, and nicotine (the internal standard) from Eastman Kodak. Stock solutions of 20 mM caffeine and 16 mM nicotine in distilled deionized water are prepared in advance. The tea was Lipton choicest blend orange pekoe and pekoe cut black; the coffee was Chase and Sanborn. Preparation of Samples and Standard Solutions A 900-µL aliquot of each soft drink is mixed with 100 µL of the internal standard solution. The tea and coffee beverages are prepared according to manufacturer’s recommendations and diluted 1:4 with distilled deionized water. Each sample is injected in triplicate. Standard solutions of caffeine representing a concentration range of 17–370 mg/L are also prepared by dilution of the stock solution with distilled deionized water. A 900-µL aliquot of each standard solution is added into separate 2-mL vials containing 100 µL of the internal standard solution.
Instrumentation A potential of 25 kV (70 µA) is applied to a 100 cm 3 75 µ i.d. length of fused silica capillary (Polymicro Technologies, Phoenix, AZ) by a 0–30 kV dc power supply (Spellman Model RHR30PN30, Plainview, NY). The anodic buffer solution is protected in a wooden housing (36 cm 3 30 cm 3 25 cm) that contains a safety relay switch connected to a power supply (BK Precision, Chicago), which disconnects the potential when the top cover *Corresponding author.
Figure 1. Schematic illustration of CE apparatus.
Vol. 73 No. 12 December 1996 • Journal of Chemical Education
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In the Laboratory
Table 1. Milligrams of Caffeine per 12-oz (355-mL) Serving Beverage
Figure 2. Electropherogram of Pepsi.
Procedure The buffer solution consists of 50 mM sodium borate adjusted to pH 8.5 with phosphoric acid. The solution is filtered through a 0.2-µ membrane filter (Alltech, Deerfield, IL), then placed in both the anodic and cathodic reservoirs (150-mL beakers). Before any sample analysis, the capillary is rinsed with 0.1 N NaOH for 5 min using an HPLC pump (Perkin–Elmer Series 100). Buffer solution is then pumped through the capillary for 10 min. Between sample injections the capillary may be rinsed with buffer solution for 5 min. Results and Discussion An electropherogram of Pepsi appears in Figure 2. It is representative of the separation profiles of the beverages selected. The migration times of caffeine and the internal standard are 14.1 min and 15.6 min, respectively, under the experimental conditions reported here. Other species present in the beverages are not detected, rendering the procedure highly selective. A linear calibration curve for five standard solutions ranging from 17 mg/L to 370 mg/L of caffeine is obtained and may be expressed as log R = log K + n log C where R is the ratio of area responses of caffeine to the internal standard, K is the slope of the curve (sensitivity), and C is the concentration of caffeine. When n is equal to 1 ± 5% it is considered to be in the linear range (12). Caffeine levels in the beverages examined range from 27.0 to 164 mg per 12-oz (355-mL) serving (Table 1). The amount of caffeine in soft drinks determined by the CZE procedure outlined here compares favorably with the levels reported elsewhere (9–11). In evaluating the feasibility of this CZE experiment, the detection limit was estimated by determining the signal to peak-to-peak noise ratio (S/N) of the lowest caffeine standard (17 mg/ L) and multiplying the concentration of the standard by 3 (three times the noise level), then dividing by the measured S/N. The detection limit was calculated to be 1.9 mg/L, or 680 µg caffeine per 12-oz (355-mL) serving. There was no evidence of caffeine present above the detection limit in Sprite or caffeine-free Diet Coke. In addition to introducing a student to an emerging powerful analytical technique, this experiment offers sev-
1170
mga
mgb
mgc
mgd
Coke
27.0 ± 1.6
46
24.7
39.3
Diet Coke
43.9 ± 2.5
46
32.7
47.7
Pepsi
31.4 ± 1.6
38
37.6
38.2
–
39.2
–
Mountain Dew
39.0 ± 1.7
Sprite
*
Caffeine-Free Diet Coke
*
Tea
110 ± 15
Coffee
164 ± 17
*Denotes below estimated detection limit of 675 mg per serving. aAverage of triplicate determinations ± relative standard deviation using CZE procedure. bConsumer Reports 1991, 56, 525. c Reference 9 using HPLC. dReference 11 using HPLC.
eral other attractive features. The results of caffeine determinations by CZE may be compared in another laboratory period with the corresponding caffeine data generated by one of the complimentary HPLC experiments published in this Journal (9–11). Detection limits, precision, and linear dynamic range of the two techniques are comparable (13). Furthermore, the numerous readily available brands of coffee and tea provide a convenient supply of samples. The following listing of vendors of complete capillary electrophoresis is presented for the convenience of those considering commercial instrumentation (14): ATI Unicam; Beckman Instruments, Inc.; Bio-Rad Laboratories; Bischoff Analysentechnik und -gerate GmbH; Dionex Corp.; Government Scientific Source; HewlettPackard Company; Isco Inc.; Jones Chromatography; Kontron Instruments SpA; Lauerlabs; Perkin–Elmer Corp.; Sci-Con; Stagroma AG; Thermo Separation Products; Waters Corp. Acknowledgments We are grateful to Martin Fuchs of Waters for his help in the modification of the HPLC detector and to Vern Rheinhold of Harvard University for providing the power supply and detector. Note 1. Present address: National Center for Toxicological Research, Jefferson, AR 72079.
Literature Cited 1. Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298. 2. Heiger, D. N. High Performance Capillary Electrophoresis—An Introduction; Hewlett-Packard Co. Publication No. 12-5091-6199E, 1992; p.7. 3. Li, S. F. Y. Capillary Electrophoresis—Principles, Practice and Applications; Elsevier: Amsterdam, 1992. 4. Ewing, A. G.; Wallingford, R. A.; Olefirowicz, T. M. Anal. Chem. 1989, 61, 292A. 5. The Methylxanthine Beverages and Foods: Chemistry, Consumption and Health Effects; Spiller, G.A., Ed.; Liss: New York, 1984. 6. Dews, P. B. Caffeine: Perspectives from Recent Research; Springer–Verlag: Berlin, 1984. 7. Yoshikura, H. Nature 1974, 252, 71. 8. Rothwell, K. Nature 1974, 252, 69–70. 9. Delaney, M. F.; Pasko, K. M.; Mauro D. M.; Gsell D. S.; Korolog, P. C.; Morawski J.; Krolikowski, L. J.; Warren, F. V. J. Chem. Educ. 1985, 62, 618. 10. DiNunzio, E. D. J. Chem. Educ. 1985, 64, 446. 11. Strohl, A. N. J. Chem. Educ. 1985, 64, 447. 12. Dressler, M. Selective Gas Chromatographic Detectors; Elsevier: Amsterdam, 1986. 13. Stevenson, R. Am. Lab. 1993, 25(5), 52. 14. LC*GC 1994, 12, 608.
Journal of Chemical Education • Vol. 73 No. 12 December 1996
