In the Laboratory
The Determination of Methylxanthines in Chocolate and Cocoa by Different Separation Techniques: HPLC, Instrumental TLC, and MECC Abel Carlin-Sinclair* and Iman Marc Département de Chimie, Université de Versailles-Saint-Quentin-en-Yvelines, Bâtiment Descartes, 45 avenue des Etats Unis, 78035 Versailles, France; *
[email protected] Laurence Menguy and Damien Prim Université de Versailles-Saint-Quentin-en-Yvelines, Institut Lavoisier–UMR CNRS 8180, Bâtiment Lavoisier, 45 avenue des Etats Unis, 78035 Versailles, France
The determination of caffeine in coffee or other beverages by high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE) is a popular topic in educational analytical chemistry (1–7). Studies to analyze the three methyl xanthines (caffeine, theobromine, and theophylline; Figure 1) in cocoa products using either HPLC (8–11) or micellar electrokinetic chromatography (MECC) (7, 12, 13) have also been reported. As a culmination of an analytical course1 we ask students to separate the three structurally similar methylxanthines from commercially available chocolate or cocoa. Our approach is based on the unique comparison of the results obtained by three separation techniques after solid-phase extraction (SPE): HPLC, MECC, and instrumental thin-layer chromatography (I-TLC). These methods differ with respect to separation mode and provide orthogonal information concerning related substances and impurities. Along with the aforementioned pedagogic reasons, an additional motivation to consider chocolate and cocoa as substrates for this study was to connect scientific studies to daily life consumption products and historical scientific knowledge. The “marvelous history” of chocolate and cocoa has been nicely reviewed by Tannenbaum and others (14) from the discovery of this natural product to the transformation processes leading to current common uses and packaging modes, highlighting the chemical composition, the chemical and physical modifications as well as flavor and textural changes during processing.
O H3C O
O H3C O
H N
N
CH3 N
N N
N
O
CH3
N CH3
theophylline
N
N
HN
caffeine O
CH3
N
N
CH3
theobromine
Figure 1. Structural formulas of three methylxanthines found in chocolate and cocoa. Note that the structures differ in the number and position of the methyl groups.
Experimental Details Chemicals and Equipment Analytical-grade sodium dodecyl sulfate (���������� SDS), caffeine, theobromine, and theophylline were obtained from Fluka–Aldrich (L’Isle d’Abeau Chesnes, Saint Quentin Fallavier, France). Deionized water was prepared with a Milli-Q system (Millipore, Saint Quentin, France). HPLC-grade methanol and ethyl acetate were obtained from SDS (ZI de Valdonne, 13124 Peypin, France). Phosphate salts were obtained from ACROS (Noisy-le-grand, France). The chocolate bar (Lindt 70%) and cocoa powder (Suchard) were obtained from a local supermarket. An SPE vacuum system, an HPLC–UV system with a C18 column, a CE–UV system, and an I-TLC system were used. (Details are available in the online material.) Procedures The experiment is run over four, half-day periods as follows:
• SPE extraction of methylxanthines from chocolate and cocoa and preparation of calibration standards.
• Optimization of the separation by HPLC of the three standards, preparation of calibration curves, and quantification of samples.
• Optimization of the separation by TLC of the three standards, preparation of calibration curves, and quantification of samples by I-TLC.
• Optimization of the separation by MECC of the three standards, preparation of calibration curves, and quantification of samples. Comparison and co-validation of the three techniques.
SPE Extraction of Methylxanthines from Chocolate and Cocoa Natural products are usually complex mixtures containing a multitude of chemical components. The first step was to selectively extract the methylxanthines from the solid matrix. In this educational context, SPE represents an adequate technique to selectively elute the methylxanthines from the complex matrix. Samples were obtained by dissolving 500 mg of freshly crushed chocolate or cocoa powder in 20 mL Milli-Q water. The solutions were placed in sealed tubes and heated in a water bath at 80 °C for 15 min. Sample extracts were obtained
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 86 No. 11 November 2009 • Journal of Chemical Education
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In the Laboratory
through sequential-conditioning of the SPE tube, addition of the sample, washing, and several elutions (Figure 2). The steps are outlined as 1. Condition the SPE tube in vacuum(5 mm Hg) Activation: 1 mL methanol Equilibration: 1 mL Milli-Q water
2. Sample loading: 0.5 mL of sample
3. Wash the packing: 1 mL Milli-Q water
4. Elute the compounds of interest: 2 times with 2.5 mL of meth anol in separate vials (slow or dropwise flow rate is beneficial)
5. Evaporation: The 2 vials were concentrated to dryness under nitrogen at 40 °C and each residue was dissolved in 1000 μL of Milli-Q H2O and then separated in 5 aliquots of 200 μL. Three of the aliquots were analyzed by HPLC, MECC, and I-TLC.
6. Reconstituted for each analytical technique: Each analytical technique uses different mobile phases, so students have to prepare the extracts with different proportion of solvents for each technique.
Preparation of Standards Solutions The standards were prepared for each analytical method. For quantification five mixed-standards solutions were prepared from 10–5 mol L–1 to 5 × 10–4 mol L–1. Optimization of the Separation by HPLC of the Mixed-Methylxanthines Standards and Samples Students optimized the mobile phase with methanol and water on a C18 stationary phase. Using a mixed-methylxanthine solution, the first mobile phase tested was methanol. The
activation & equilibration
sample loading
washes
elution 1
solvent elution strength was then weakened until separation of all methylxanthines occurred. Elution order was discussed and confirmed by comparison with separate injection of each methylxanthine. Characteristic separation parameters such as efficiency (N), retention factor (k), and selectivity (α) in the determined conditions were measured. Calibration solutions were injected, from the lowest to the highest concentration. For the quantification of each methyl xanthine in extracts, calibration curves were set up, plotting the peak areas of each standard measured at UV wavelength of 272 nm versus the concentration of mixed-standards solutions. Each extract solution was injected twice and the calculated average area value accounted for quantification. Optimization of the Separation by TLC of the Three Methylxanthines Standards and Samples The separations were realized on classical silica gel plates. With the CAMAG twin trough chambers, 6 mL of solvent was required for the development of a 5 ������������������������� cm × 10 �������������������� cm plate. ������� Optimization of the mobile phase was achieved stepwise: optimization of the mobile-phase strength ε° followed by optimization of efficiency parameter, N. In the first step students modified the ratio of given solvents (MeOH and ethyl acetate) starting from a 1:1 ratio until satisfactory separation occurred. The objective of the second step was to reduce the thickness of spots, w, which is essential for optimal quantification in I-TLC. Students added aqueous NH3 (30%) to the I-TLC chambers in various ratios to the aforementioned MeOH∙ethyl acetate combination. Students justified their choice after analysis of migrations: Rf values and spots aspect. In I-TLC, students analyzed both standards and samples on the same plate under identical migration conditions. Up to
elution 2
HPLC
evaporation reconstitution MECC
N2
TLC
analytes interferences
Figure 2. SPE extraction procedure.
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Journal of Chemical Education • Vol. 86 No. 11 November 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Laboratory A
Methanol is flammable, is toxic by ingestion, and is a skin and eye irritant. Ethyl acetate is flammable and causes irritation to skin, eyes, and respiratory tract. SDS is harmful by inhalation and is irritating to eyes, skin, and respiratory system. The ammonia, hydrochloric acid, and sodium hydroxide solutions are corrosive. Gloves and safety glasses were used during the preparation of the solutions. Results and Discussion Optimization of the Separation The retention times and separation of theobromine, theophylline, and caffeine by HPLC are shown in Figure 3A. Optimal results were obtained using MeOH∙H2O 30∙70 v/v as the mobile phase. The phosphate buffer 20 mM at pH 8 and SDS 20 mM were used for the optimal separation via MECC of theophylline, theobromine, and caffeine, and their injection alone led to a clear identification of each compound (Figure 3B). Optimization of the mobile phase [AcOEt∙MeOH∙NH3 (90∙10∙8.9 v/v/v] led to fair separation by I-TLC of theophylline, theobromine, and caffeine (Figure 3C). Elution, on the same TLC plate, of standards of theobromine, theophylline, and caffeine led to a clear identification of each compound. In each separation technique, selective interactions such as hydrophobicity, adsorption, dipole–dipole interaction, or solubility in micelles account for the separation. In their report students must justify the migration order and observed differences among HPLC, I-TLC, and MECC (Table 1).
theophylline
0
1
2
3
4
5
6
Time / min
Detector Response
B 3 standards theobromine theophylline caffeine 0
1
2
3
4
5
Time / min C 3 standards
Detector Response
Hazards
theobromine
caffeine
Optimization of the Separation by MECC of the Three Methylxanthines Standards and Samples Methylxanthines are neutral compounds, as a consequence the separation was realized under MECC technique using a silica capillary. Generation of micelles was achieved using SDS. Optimization of the separation required the following instrumental parameters: injection of 0.3 psi during 2 s and separation of 20 kV charge. The conditioning sequence was completed by applying the buffer electrolyte for 5 min. Students optimized the latter starting from phosphate buffer 20 mM at pH 8 and SDS 10 mM by increasing the SDS concentration until complete separation. Calibration solutions were injected, from the lowest to the highest concentrations. ������������������������������������� C������������������������������������ alibration curves were set up, plotting peak areas measured at UV wavelength of 280 nm of each standard versus the concentration of mixed-standards solutions. Each extract solution was injected twice and the calculated average area value accounts for quantification.
3 standards
Detector Response
32 tracks can be used in a classical 20 cm ×10 cm plate without any interference, allowing the alternative deposit of calibration solutions and samples on the same plate. In our case, each sample was deposited twice. A volume of 15 mL of the optimized mobile phase was used for migration with the twin trough chamber for a 20 cm × 10 cm plate. All tracks were scanned at 272 nm using the CAMAG TLC Scanner3. Integration of the area of each spot and evaluation of the concentration of each component of samples were realized using CATS 4.06 program.
theophylline
theobromine caffeine 20
30
Distance / mm
40
50
Figure 3. Separation and identification of methylxanthines in optimal conditions: (A) HPLC, (B) MECC, and (C) I-TLC.
Table 1. Migration Order of Methylxanthines Migration Order
HPLC
MECC
1
theobromine
theobromine
caffeine
2
theophylline
theophylline
theobromine
3
caffeine
caffeine
theophylline
I-TLC
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 86 No. 11 November 2009 • Journal of Chemical Education
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In the Laboratory
Quantitative Analysis Two different sources—chocolate and cocoa—of theobromine, theophylline, and caffeine have been quantified in this study. Results of the quantitative analysis of two extracts per each sample are shown in Table 2. It is worth noting that similar values were obtained regardless of the technique used. For both the chocolate and the cocoa extracts, theophylline could not be detected with certainty, the supposed peaks approaching the limit of detection. According to literature, only traces of theophylline are present in cocoa and chocolate (8, 9). The values determined by the students have to be analyzed to co-validate the different techniques. These results may also be compared with literature average values obtained for other studies (9–13, 15–17).
Table 2. Mass Percent of Methylxanthines in Chocolate and Cocoa Samplea
Cocoa
Chocolate
Ingredient
Methylxanthine (mass %)b HPLC
MECC
I-TLC
theobromine
2.03
2.04
1.96
theophyllinec
—
—
—
caffeine
0.17
0.14
0.17
theobromine
1.18
0.96
1.02
theophyllinec
—
—
—
0.11
0.09
0.09
caffeine a
Two injections or deposits of two extracts per sample (n = 4). b Mass % is the mass of methlyxanthine per mass of the sample. c Uncertain results because at the limit of detection.
Conclusion The quantitative determination of structurally related caffeine, theobromine, and theophylline using “orthogonal separation techniques”, HPLC, MECC, and I-TLC, is described. This experiment is suitable for students over four, half-day periods. The conclusions in the student reports validate all techniques. Gratifyingly consistent results were obtained among these techniques regardless of the separation modes in both chocolate and cocoa. Acknowledgments The authors gratefully thank the Institut Supérieur International du Parfum de la Cosmétique et de l’Aromatique Alimentaire (ISIPCA), Aldrich, and the Chemistry Department of the Université de Versailles-Saint-Quentin-en-Yvelines for technical and financial support. Note 1. This lab is used as a practical at the master level at the end of an alimentary flavor course. This practical addresses analytical chemistry and separation techniques.
Literature Cited 1. Beckers, J. L. J. Chem. Educ. 2004, 81, 90–93. 2. Van Atta, R. E. J. Chem. Educ. 1979, 56, 666. 3. DiNunzio, J. E. J. Chem. Educ. 1985, 62, 446–447. 4. Strohl, A. N. J. Chem. Educ. 1985, 62, 447–448. 5. Ferguson, G. K. J. Chem. Educ. 1998, 75, 467–469. 6. Conte, E. D.; Barry, E. F.; Rubinstein, H. J. Chem. Educ. 1996, 73, 1169–1170. 7. Vogt, C.; Conradi, S.; Rohde, E. J. Chem. Educ. 1997, 74, 1126–1130.
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8. Brunetto, M. R.; Gutierrez, L.; Delgado, Y.; Gallignani, M.; Zambrano, A.; Gomez, A.; Ramos, G.; Romero, C. Food Chemistry 2007, 100, 459–467. 9. Tokusoglu, O.; Unal, M. K. Eur. Food Res. Technol. 2002, 215, 340–346. 10. Caudle, A. G.; Gu, Y.; Bell, L. N. Food Research Int. 2001, 34, 599–603. 11. Rojo de Camargo, M. C.; Toledo, M. C. F. J. Sci. Food Agric. 1999, 79, 1861–1864. 12. Gotti, R.; Fiori, J.; Mancini, F.; Cavrini, V. Electrophoresis 2004, 25, 3282–3291. 13. Pomilio, A.; Trajtemberg, S.; Vitale, A. J. Sci. Food Agric. 2005, 85, 622–628. 14. Tannenbaum, G. J. Chem. Educ. 2004, 81, 1131–1135; and references cited. 15. Hershey’s Chocolate Products Nutritional Information. http:// www.hersheys.com/nutrition/caffeine.asp (accessed Apr 2009). 16. Cacao Barry Home Page. http://www.cacao-barry.com (accessed Apr 2009). 17. Kunugi, A.; Tabei, K. J. High Resol. Chromatogr. 1997, 20, 456–458.
Supporting JCE Online Material
http://www.jce.divched.org/Journal/Issues/2009/Nov/abs1307.html Abstract and keywords Full text (PDF) with links to cited URLs and JCE articles Supplement Detailed instructions for the students Detailed notes for the instructor Description of the instruments
Journal of Chemical Education • Vol. 86 No. 11 November 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
