In the Laboratory
Quantitative Determination of Caffeine in Beverages Using a Combined SPME-GC/MS Method Min J. Yang, Maureen L. Orton, and Janusz Pawliszyn* The Guelph-Waterloo Center for Graduate Work in Chemistry, University of Waterloo, Waterloo, ON N2L 3G1, Canada Caffeine occurs naturally in tea, coffee, and cola nuts. Caffeine analysis is performed for quality control purposes, for example to insure proper caffeine levels in decaffeinated coffee to meet regulatory standards. Current methods for determining caffeine in beverages require pH adjustments and often involve the use of toxic organic solvents. These methods are labor-intensive, generate large quantities of organic waste, and have relatively poor precision. Alternatively, solid-phase microextraction (SPME) can be used. The SPME technique has many advantages including simplicity, portability, time efficiency, sensitivity, and compatibility with a GC system. Initial work on SPME caffeine analysis has been reported (1). The SPME principle is based on an equilibrium process in which the analyte partitions between the fiber and the aqueous phase (2, 3). Many applications of SPME have been investigated (4–8), and a recent review provides a good overview (9). The main objective of this paper is to describe a simple undergraduate experiment for determination of caffeine in common beverages using the combined SPME-GC/MS methods. Experimental Procedure
Materials and Equipment The SPME device was purchased from Supelco (Supelco Canada, Mississauga, ON). For caffeine analysis, an uncoated fused silica fiber was used for extraction. Commercial fibers are often supplied with a coating such as poly(dimethylsiloxane) (PDMS) and polyacrylate. Uncoated fiber can be prepared by dissolving the fiber coating in hot sulfuric acid. Two 5-mg/mL stock solutions of regular 12C caffeine (Aldrich Chemical Co, Inc., Milwaukee, WI) and isotopically labeled (trimethyl 13C) caffeine (Cambridge Isotope Laboratories, Woburn, MA) were separately prepared in methanol. Regular coffee, decaffeinated coffee, and teas were brewed as for normal consumption. Soft drinks were taken from beverage containers. A Varian 3400 gas chromatograph equipped with a Saturn II ion trap MS (Varian, Mississauga, ON) detector was used. The column was a 30 m × 0.25 mm SPB-5 with a stationary phase thickness of 0.25 µm (Supelco Canada, Mississauga, ON). Extraction Procedures Many different versions of SPME procedures are described; which one to use depends on the application (4–8). For this experiment, the adsorption period was set for 5 minutes. The sample was magnetically stirred during extraction (Fig. 1). After the adsorption step, the fiber was directly transferred into the GC injector in which the analytes were thermally desorbed at 250 °C, and the GC/MS run was started. To obtain a complete desorption, the fiber *Corresponding author.
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Figure 1. Sample extraction setup for the manual SPME method.
was allowed to stay in the hot GC injection port for 1 min before withdrawal.
GC/MS Methods The GC oven was maintained at 200 °C. for 1.5 min, then ramped at 30 °C/min to 275 °C. The temperatures of the injector and transfer line were set at 250 and 220 °C, respectively. Helium was used as the carrier gas at a flow rate of 1.0 mL/min. MS conditions were electron impact, ionization, positive ion, mass range 50–300 amu, and 60 scans/min. The integrated areas from the extracted mass chromatogram of the molecular ions (194 and 197 for 12C caffeine and 13C 3 caffeine) were used for all quantitation. The entire caffeine analysis run took 4 minutes to complete.
Determination of the Calibration Curve For quantitative caffeine determination, a calibration curve was obtained by analyzing a set of standard aqueous samples with 12C caffeine concentrations of 50, 100, 200, 300 and 500 µg/mL spiked with 240 µL of the 13C 3 caffeine stock solution, which served as an internal standard. The ratio of the integrated peak areas for 12C caffeine (m/z 194) and 13C3 caffeine (m/z 197) was plotted versus 12C caffeine concentration in the standard solutions to provide external calibration for caffeine determination in various beverage samples. This method of isotopic dilution has been previously described (10).
Analysis of Caffeine Content in Selected Beverage All beverages were allowed to reach room temperature before the analysis. A 12-mL aliquot of each beverage was
Journal of Chemical Education • Vol. 74 No. 9 September 1997
In the Laboratory Table 1. Quantitative Analysis of Caffeine in Beverages Using the SPME-GC/MS Method Peak ratio (m / z 194/197)
Beverage
Caffeine conc. (µg/mL)
100% Colombian coffee
5.6332
635
Decaffeinated coffee
0.4468
49 206
Chinese green tea
1.8315
Camomille tea
0.6596
73
Diet Coke
2.311
260
Diet decaffeinated Coke
0.4769
52
pipetted into a 15-mL vial. To each sample vial, 240 mL of the 13C3 caffeine stock solution was added as an internal standard. The ratio of the integrated peak area for both 12C caffeine (m/z 194) and 13C3 caffeine (m/z 197) was used together with the previous calibration data to quantitatively determine the caffeine content in the beverage. Table 1 shows the calculated average caffeine concentration for the tested beverages based on the standard calibration curve. Results and Discussion For caffeine extraction from beverages, an uncoated fiber is preferred because caffeine contents in the beverages of interest are in the ppm range. Also, uncoated fiber minimizes potential carry-over problems and produces sharp injection bands owing to rapid desorption. Alternatively, the 7-µm PDMS fiber can be used, but in this case the carryover may become a significant issue. Since the internal standard and native analyte have the same chemical and physical properties, their behavior during extraction (equilibration time, metric effects) is identical. As a result, the use of an isotopically labeled internal standard essentially eliminates the need to reach equilibrium of analyte partitioning between the fiber and the sample. Also, any change in extraction conditions, including the change of the fiber properties due to irreversible adsorption of some of the matrix component, is compensated for. Quantitative information can be obtained by generating extracted mass chromatograms for the specific ions of the native analyte and the isotopically labeled internal
Retention Time (s)
Figure 2. Typical chromatograms obtained for caffeine analysis of a brewed coffee sample using the SPME-GC/MS method. (a) A total ion chromatogram. (b) An extracted mass chromatogram for m /z 194. (c) An extracted mass chromatogram for m/ z 197.
Figure 3. A standard calibration curve for quantitative caffeine determination using the SPME-GC/MS method.
standard, and comparing the two peak areas obtained. Figure 2a shows that both the isotopically labeled (trimethyl 13C) caffeine internal standard and the native 12C caffeine coelute at the end of the GC column and form a single caffeine peak in the total ion chromatogram. Figures 2b and 2c show the extracted mass chromatograms for the molecular ions of the native caffeine (m/z 194) and the 13C 3 caffeine (m/z 197), respectively. There is a linear relationship between the amount of caffeine adsorbed by the bare fiber and the concentration in the solution. The precision of the method in terms of relative standard deviation (RSD), determined by analyzing 5 vials of the same sample, is 1.9%. A calibration curve (Fig. 3) was obtained by plotting the peak area relations between the 12C caffeine and the 13C 3 caffeine versus the 12C caffeine concentration. Excellent linearity can be observed for the SPME-GC/MS method within the tested concentration range. The correlation coefficient for the linear regression was .996. The quantitative results are in good agreement with caffeine contents reported by Hawthorne et al. (1). There was concern that deposition of beverage color stain on the fiber surface could hinder sample extraction. For replicate sample analysis, large fluctuations in component peak areas were observed between experimental runs under the same conditions. These fluctuations were effectively compensated by the internal standard method and the peak area ratio for the replicate samples remained relatively unchanged. In other words, use of the internal standard has avoided this uncertainty and leads to reproducible quantitative results. The initial acquisition of the GC/MS system and its maintenance would be the most expensive part of the experiment. The other costs can be considered minimal. The SPME device and extraction fiber can be purchased directly from Supelco for ~$200 U.S. and can be reused many times. Only a small amount of (trimethyl 13C) caffeine is needed for the experiment; for instance, 1 g of 13C-labeled caffeine costing ~$200 U.S. is sufficient to spike more than 800 samples. If the instruments are set up and stock solutions are prepared for the students, the entire experiment can be completed within three hours. The instructor may choose to prepare and run one set of calibration standards each day for all groups of students so that the students will have more time to comprehend the operation of the instrument and to analyze their data. A group of no more than four students should be allowed to run the experiment at one time to ensure sufficient exposure to the use of the instruments. Each student should be given an unknown sample to run.
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In the Laboratory The instructor may also choose to give students real or simulated beverage samples as unknowns. A simulated beverage sample can be prepared by making an aqueous caffeine solution of any concentration, with or without dye addition.
GC/MS method for quantitative determination of caffeine in beverages has been implemented as a second-year undergraduate laboratory experiment at the University of Waterloo, Waterloo, Ontario. We would like to thank the undergraduate laboratory TA, Lin Pan, for her cooperation in sharing the instrument.
Conclusions Literature Cited SPME is a fast, inexpensive, and solvent-free alternative for extracting organic compounds from sample matrices. Recent publications (4–8) have revealed its wide acceptability and applicability in many environmental and industrial applications. The technique certainly deserves a place in the classroom. The SPME-GC/MS method for analysis of caffeine in beverages is an appropriate hands-on experiment post-secondary level instrumental laboratories. The experiment will intrigue students learning the principles of analytical instrumentation and sample preparation techniques because the experiment demonstrates the use of today’s technology in a simple fun-to-do real life application. Acknowledgments This work has been financially supported by the Natural Sciences and Engineering Research Council of Canada, Supelco Canada, and Varian Canada. The combined SPME-
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