Caffeinated Beverages - American Chemical Society

Chapter 40

Capillary Electrophoresis of Some Caffeinated Soft Drinks Jennifer M. Ames, Louise Royle, and H a r r y E. Nursten

Downloaded by UNIV LAVAL on July 13, 2016 | http://pubs.acs.org Publication Date: January 15, 2000 | doi: 10.1021/bk-2000-0754.ch040

Department of Food Science and Technology, The University of Reading, Whiteknights, Reading RG6 6AP, United Kingdom Capillary electrophoresis (CE) is a family of techniques which shows tremendous promise for the analysis of beverages. The very high resolving power, short analysis time, low sample requirement, sensitivity and ease of automation associated with C E make it ideal for the analysis of aqueous samples, such as soft drinks. Capillary zone electrophoresis (CZE), using carbonate buffer at p H 9.5 and detection at 200 and 280 nm, was applied to different cola samples. Caramel, caffeine, acesulfame K , aspartame, saccharin and sodium benzoate all migrated within 12 minutes. Seven of the non-diet and 12 of the diet colas contained high-sulfur, high-nitrogen Class IV caramel at concentrations estimated to be between 0.57 and 0.82 g solids per litre.

Capillary electrophoresis (CE) comprises a group of separation techniques that have attracted considerable interest over the last ten years for the analysis of aqueous samples. Separations are based, for example, on the charge:mass ratio in free solution (capillary zone electrophoresis, C Z E ) , interactions between micelles and solutes (micellar electrokinetic chromatography, M E K C ) , isoelectric point (capillary isoeiectrofocusing, cIEF), size (dynamic sieving electrophoresis, DSE) or partitioning between stationary and mobile phases (capillary electrochromatography, CEC). The simplest and most commonly used method is C Z E . The advantages of C E over H P L C include very high separation efficiency, typically 4 000 000 theoretical plates per meter, compared to around 100 000 plates per meter for an H P L C column (/), nanoiitersize injection, separation in an aqueous medium and short run time. Detection at 200 nm, often with increased sensitivity, is possible because no organic solvent in present is the buffer, allowing detection of analytes with weak chromophores. According to the legal definition, soft drinks include fruit drinks (but not fruit juices), soda water, tonic water and artificially carbonated water, whether flavored or unflavored, ginger beer and herbal beverages (2). They are aqueous 394

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solutions or dispersions of a selection of components, including flavorings, sugars, artificial sweeteners, other compounds contributing to taste (e.g., caffeine, acidulants), preservatives and coloring materials (e.g., azo dyes, carotenoids and caramel). Most of the major ingredients of soft drinks can be determined easily by H P L C (J), but some components, notably caramels, present an analytical challenge. The simultaneous determination of components present in soft drinks by H P L C is sometimes possible (4). For example, a reversed-phase method has been used to separate acesulfame-K, aspartame, saccharin, sorbic acid, benzoic acid and caffeine in less than 15 minutes (5). H P L C methods for the separation of synthetic food colors have not so far included the simultaneous analysis of other components such as sweeteners and preservatives (3). C E offers the possibility of separating most, i f not all, of the components of an individual soft drink within one run. Several C E methods have been published for the simultaneous analysis of sweeteners and preservatives (6-10), while different, but related, methods have been published for the determination of preservatives and synthetic colors (11,12). Most previous studies on the C E separation of soft drink components have used borate buffer at pH 9.4-10.0 (7, ΙΟ Ι 2) and, e.g., acesulfame K , aspartame, benzoic acid, methyl, ethyl and propyl 4hydroxybenzoates, saccharin and sorbic acid can all be separated within 9 minutes (7). Good separation, within 12 minutes, of a wider range of sweeteners (acesulfame K , alitame, aspartame, dulcin and saccharin), as well as caffeine, benzoic acid and sorbic acid, has been achieved by M E K C , involving p H 8.6 borate/phosphate buffer containing sodium deoxycholate (8). Recently, we described a C Z E method for the identification and quantitation of Class IV caramels in soft drinks (13). This paper reports the separation of other major declared ingredients of colas and related drinks, using the same conditions of analysis.

Experimental Class IV caramels were obtained from two British manufacturers. Quinoline yellow and brilliant blue were obtained from Pointings Ltd (Prudhoe, U K ) . Sucrose was from a local supermarket, acesulfame Κ was from Hoechst, Germany, and aspartame, caffeine, saccharin and sodium benzoate (all the purest grade available) were from Sigma (Poole, U K ) . A total of 55 soft drinks was obtained from both supermarkets and local shops. They comprised colas colored with caramel, other caramel-containing drinks, and drinks sold as 'green cola', i.e., colored with quinoline yellow and brilliant blue, instead of caramel. The colas colored with caramel could be placed into eight categories (A-H), according to the major declared ingredients they contained. Four further categories were used for the other caramel-containing drinks (I, J) and for the green colas (K, L ) , as shown in Table I. Solutions of caramel were prepared in water. A l l samples were 0.2 μιη filtered before analysis.



G H I J K

E F

A Β C D

Category

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

Caffeine

X

Caramel

χ

χ χ χ

χ

χ

Sugar

χ

χ

χ χ χ χ χ χ χ χ χ χ

Major Declared Ingredients Acesulfame Κ Saccharin Aspartame

Table I Categorization of colas


χ χ

χ χ χ χ χ χ χ

Sodium benzoate

χ χ

Quinoline yellow

χ χ

Brilliant blue

0\

397 The C E method has been described (13). Briefly, samples were separated by C Z E using 50 m M carbonate buffer at pH 9.5 and a 48.5 cm (40 cm to the detector), 50 μιη i.d., x3 bubble capillary. Detection was at 200 and 280 nm. The instrument was a Hewlett-Packard HP C E equipped with a diode array detector and an H P C E ChemStation. 3 D


Results and Discussion Our preliminary studies compared four buffers over the p H range 9.5 to 11.0, i.e., borate, phosphate, glycine and carbonate (13). Borate and phosphate resulted in longer migration times for the colored caramel peak, due to complexation with buffer components. Carbonate gave clearer e-grams than glycine with all the major declared ingredients, apart from caramel, being separated within 8 minutes, and a total run time for samples containing caramel of 12 minutes. E-grams of the caramel standard and of a cola sample from category A , with detection at 200 nm, are given in Figure 1. The two traces are strikingly similar, the main difference being the presence of caffeine at about 3.2 min in the cola. The other major declared ingredients were also separated using the described method. Examples of e-grams with monitoring at 200 nm for samples from categories B , C and J are shown in Figure 2. Saccharin, acesulfame Κ and sodium benzoate all absorb strongly at 200 nm and migrate with the caramel peak (when present), reducing the accuracy of quantitation. Monitoring at 280 nm (Figure 3) reduced the size of the peaks due to the artificial sweeteners and preservatives, thus improving the accuracy of quantitation of the caramel. More work is needed to establish the optimum conditions for separation of all these components (which ideally should be completely resolved) in caramel-containing soft drinks. When complete resolution is not possible, quantitation of individual compounds may be improved by using a wavelength at which co-migrating components have minimal absorbance. Using the same C E conditions of analysis, standard aspartame solutions gave a good peak shape, but aspartame gave a poor peak shape tor drink samples, including colas (see Figure 2c, for an example). Standard aspartame runs well by C Z E in other buffers, phosphate-borate mixture at pH 9 (14) or glycine at pH 9 (15). Aspartame is stable at pH values as low as 3 (4% but it has been shown that 55% of the aspartame content of a p H 2.55 diet beverage are converted to identifiable degradation products after storage for 50 weeks at 20°C (14). Also, diketopiperazine (DKP), at a level of 4-5% of the total amount of aspartame, has been reported in Diet Seven-Up® soft drink (14). In the current study, any aspartame degradation products may have co-migrated with aspartame under the chosen conditions of analysis. This requires further investigation. Brilliant blue and quinoline yellow were present in the category J sample. Brilliant blue gave a broad peak that co-migrated with aspartame and which could be a further reason for the poor peak shape for aspartame in the category J sample. Quinoline yellow gave two main peaks, at around 8 and 9 minutes. The buffer strength used is not ideal for these synthetic dyes which give sharper peaks at lower buffer strengths, e.g., 15 m M (16).



Figure J. E-grams of (a) the caramel standard and (b) a category A cola (see Table I), nm in 50 mM carbonate buffer at pH9.5 with detection at 200 nm.


399

Aspartame

mAU 101


Caffeine

10

mAU

Aspartame

12

• 14 mm

Sodium Benzoate

25

20 Caffeine

Acesulfame Κ

15

10

Β 10

1 2

min

1 4

Figure 2. E-grams of cola samplesfrom(a) category B (b) category C and (c) category J (see Table I), run in 50 mM carbonate buffer at pH 9.5 with detection at 200 nm. f



400

4

6

8

10

12

m

i

n

14

Figure 2. Continued.

10 mAU Caffeine

Sodium Benzoate

10

12

mm

14

Figure 3. E-grams of cola samplesfrom(a) category B, (b) category C and (c) category H (see Table I), run in 50 mM carbonate buffer at pH 9.5 with detection at 280 nm.



401

mAU,

Saccharin

IS

id

Sodium Benzoate

-y—ι

6

8

10

12

Figure 3. Continued.


14 mm

402

Conclusion A reliable, robust and rapid C Z E method, based on carbonate buffer, for the determination of caramel in soft drinks shows considerable potential for the simultaneous quantitation of several other major components of soft drinks. Such a procedure should permit the determination of all components of interest within a 12 minute run, giving benefits in sample throughput and running costs.


Acknowledgments We thank the caramel producers for the caramel samples, Dr L Castle for helpful discussions, Mrs C M Radcliffe and Mrs Ε Inns for technical support, the Ministry of Agriculture, Fisheries and Food (UK) for funding the work and The University of Reading and The Royal Society for travel grants.

References

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Baker, D.R. Capillary Electrophoresis; John Wiley and Sons Inc.: Chichester, UK, 1995. Green, L.F. In Developments in Soft Drinks Technology; Green, L.F., Ed.; Applied Science: London, UK, Vol. 1, 1978, pp 1-29. Nollet, L.M.L. Ed. Handbook of Food Analysis, Vol. 2, Residues and Other Food Component Analysis. Dekker: New York, 1996. Prodolliet, J. In Handbook of Food Analysis; Nollet, L.M.L. Ed.; Dekker: New York, 1996, Vol. 2, Residues and Other Food Component Analysis, pp 1835-1865. Hagenauer-Hener, U.; Frank, C.; Hener, U.; Mosandl, A. Deutsche Lebensmittel-Rundschau 1990, 86, 348-351. Jimidar, M.; Hamoir, T.P. J. Chromatogr. 1993, 636, 179-186. Schuster, R.; Gratzfeld-Hüsgen, A. Hewlett-Packard Application Note, 1994. Thompson, C.O.; Trenerry, V.C.; Kemmery, B. J. Chromatogr. 1995, 694, 507-514. Walker, J.C.; Zaugg, S.E.; Walker, E.B. J. Chromatogr. 1997, 781, 481485. McDevitt, V.L.; Rodriguez, Α.; Williams, K.R. J. Chem. Educ. 1998, 625-629. Kuo, K.-L.; Hsieh, Y.-Z. J. Chromatogr. 1997, 768, 334-341. Kuo, K.-L.; Huang, H.-Y.; Hsieh, Y.-Z. Chromatographia, 1998, 47, 249-256. Royle, L.; Ames, J.M.; Castle, L., Nursten, H.E.; Radcliffe, C.M. J. Sci. Food Agric. 1998, 76, 579-587.


14. 15.


16.

Aboul-Enein, H.Y.; Bakr, S.A. J. Liq. Chrom. & Rel. Technol. 1997, 20, 1437-1444. Walker, J.C.; Zaugg, S.E.; Walker, E.B. J.Chromatogr.1997, 781, 481485. Nevado, J.J.B.; Cabanillas, C.G.; Salcedo, A.M.C. Anal. Chim. Acta 1999, 378, 63-71.


Caffeinated Beverages - American Chemical Society

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