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¨ ITAK ˙ c TUB

**Derivative Spectrophotometric Determination of Caﬀeine in Some Beverages
**

˘ ˙ G¨ uzin ALPDOGAN, Kadir KARABINA, Sıdıka SUNGUR Yıldız Technical University, Faculty of Science and Arts, Department of Chemistry, ˙ 34210, Davutpa¸ sa, Istanbul-TURKEY

Received 27.06.2000

Caﬀeine content was determined in cola, coﬀee and tea by second and third order derivative spectrophotometry without using any separation or background correction technique and reagent. The method is based on the measurement of the distances between two extremum values (peak to peak amplitudes) in the second order (cola) and third order (coﬀee and tea) derivative spectra of the sample solution. Calibration curves were constructed for the 2.0-10.0 µg ml−1 concentration range. As a reference method, reversed phase high performance liquid chromatographic procedure was developed. Commercially available beverages were analyzed by the two methods and the results were statistically compared by using t- and F-tests. Key Words: Caﬀeine, Beverages, Derivative Spectrophotometry

Introduction

Caﬀeine is widely distributed in pharmaceutical preparations and beverages. The need for a fast and selective determination method is obvious, especially when routine determinations are required. Many analytical methods have been developed for the determination of caﬀeine and the quality control of products containing caﬀeine. For the determination of caﬀeine in beverages, various analytical techniques including titrimetry1−5 spectrophotometry3,6−13 , polarography11, GC

12−14

, and

HPLC15−21 have been reported. Derivative spectrophotometric methods have been developed for the assay of caﬀeine in some pharmaceutical preparations22−26. Titrimetric methods suﬀer the disadvantage of using a large amount of sample and undergoing interference from many redox reagents with the determination. With the chromatographic methods, the use of expensive equipment and the demand for more operator attention prevent their application in small industrial laboratories where only a few analyses are performed each day. The polarographic method requires long analysis and suﬀers interferences from electrochemical impurities present in the food sample, whereas the enzymatic technique is generally considered to be too specialized for ordinary chemical laboratory use. Spectrophotometry is a fast and simple method for caﬀeine determination, but it cannot be used in samples with complex matrices because of background correction and techniques have been proposed such as thermal decomposition, direct ultraviolet irradiation alkaline treatment and an enzymatic 295

. A 10 µm Bondapak C18 (3. diluted to 100 ml with water and ﬁltered through a dry ﬁlter paper.. a model 481 variable wavelength spectrophotometer (at 270 nm) and a computer. the sample solution was mixed for 20 min by with a magnetic stirrer. To a portion of tea 1-2 g was added 100 ml of water and it was boiled until the volume was reduced to 40-50 ml. coﬀee and tea were chosen owing to their high caﬀeine content.˘ Derivative Spectrophotometric Determination of. Experimental Apparatus For derivative spectrophotometric measurement a Philips 8740 UV-VIS spectrophotometer and 10 mm quartz cells were used.0 µg ml−1).0-10. The purpose of this work is to develop a direct and simple UV-spectrophotometric method for the determination of caﬀeine in beverages without using pre-separation or background correction procedures. coﬀee and tea samples were analyzed for their caﬀeine content and the resuts were compared with those obtained by the HPLC method.The portions of each sample were analyzed using two methods. ALPDOGAN. The ﬁrst 20 ml was discarded and the ﬁltrate was used for the analysis. Chromatographic analyses were performed using a Waters HPLC system consisting of a model 510 delivery system. Derivative spectrophotometry is now a reasonably priced standard feature of modern microcomputerized UV/Vis spectrophotometry. and for eliminating the eﬀect of baseline shifts and baseline tilts30 . Cola.5-2. The ﬂow rate was 1. All spectra were recorded from 190 nm to 350 nm with 2 nm slit width. The mixture was cooled. 296 . Materials Caﬀeine and acetaminophen (internal standard) (Do˘ gu) were used as received.. Some visible spectrophotometric methods often require tedious pre-separation techniques to remove possible interferences from colored materials. et al. Internal standard (acetaminophen) solution (100 µg ml−1 ) was prepared. Milli Q water was used. 500 nm min−1 scan speed and very high smoothing. Soluble coﬀee (1. Merck.0 g) was homogenized with 100 ml of water using a magnetic stirrer for about 5 min. Quantitative analysis was accomplished by internal standard calibration. Analytical Procedures Preparation of sample solution Samples were purchased from the local markets for analysis .5 ml min−1 (pSI=2600).9 mm x 30 cm) RP column and an isocratic mobile phase containing MeOH-water (30:70) were used.28 . method27.. HPLC grade methanol and other analytical grade chemicals were purchased from E. This solution was freshly prepared and was diluted to obtain standard solutions for the preparation of calibration curves. In order to degas the cola. Derivative spectrophotometry is a useful technique for extracting qualitative and quantitative information from spectra composed of unresolved bands29 . Caﬀeine stock solution (100 µg ml−1) was prepared by dissolving 10 mg of caﬀeine in 100 ml of water. G. The samples were then diluted to provide the necessary working concentration (2. Several cola.

0 1.0 340 350 0. Absorption spectra of (a) cola (—-).5 ABS ABS ABS 1.5-289.0 2.0 0.0 2.0 .2 nm in the second derivative spectrum for cola.0 190 200 220 240 260 280 nm 300 320 0. Preparation of Calibration Graphs The second and third order derivative absorption spectra of caﬀeine standard solutions (2.0 1.5 1.0 (c) 3.0 340 350 Figure 1.5 1.5 0.-) solutions.7 nm and 300.7 nm and 265 nm were used for coﬀee. the extrema of 246. and 286. and the extrema of 286. 297 ABS 2.0 1.5 2.0 (b) 3.1 nm were used for the quantitative determination of caﬀeine in cola. In the third order derivative spectrum. 268. (c) tea (—-).0 190 200 220 240 260 280 nm 300 320 0.5 2. Regression equations of the calibration graphs were calculated by the method of least squares.˘ Derivative Spectrophotometric Determination of.5 2. Reference: Water Sample Analysis Second and third order derivative spectra of sample solutions were recorded against water.0 ABS 1.5 0. the extrema of 286.5 0.5 1....5 0.0 2.5 1. G. In the second order derivative spectrum.5 0.5 ABS 2.0 3.5 2.5 1.0 1.5 0.0 0. and caﬀeine (.5 nm in the third derivative spectrum for coﬀee.0 340 350 3.0-300.0 (a) 3. 3. et al.0 µg ml−1) were recorded against water between 190 and 350 nm..0 190 200 220 240 260 280 nm 300 320 0.0 2.7-245.0 2... ALPDOGAN.0 2.3 nm were used for tea. Peak-to-peak measurements were made at 232. (b) coﬀee (—-).0 nm in the third derivative spectrum for tea samples.7 nm and 234.0 -10.5 1. Concentration: 6 µg ml−1 .

To overcome this diﬃculty. second and third order derivatives of the absorption spectra given in Fig. 1.19 190 200 220 240 260 280 nm 300 320 -43. First order derivative spectra of (a) cola (—-). although extremum wavelengths are not exactly same as those obtained with standard caﬀeine solutions.4 340 350 68.. 93. 3 and 4 show ﬁrst. 2. (b) coﬀee (—-). because the variable background absorptions overlapping the analyte peaks are smoother in derivative spectra..66 80 60 40 40 0 0 220 240 260 280 nm 300 320 78. coﬀee and tea extract.-) solutions.03 60 (c) 40 1D 20 0 -20 -40 -60.. As seen. and caﬀeine (. Reference: Water 298 1D . et al.03 60 40 20 0 -20 -40 -60. Figs. Results and Discussion A UV-spectrophotometric method cannot be used directly for the determination of caﬀeine in beverages owing to the matrix eﬀect of UV-absorbing substances in the sample matrix. 1.35 190 200 78. Concentration: 6 µg ml−1 . In contrast.19 340 350 -62..23 (a) 40 68. matrix eﬀects are eliminated by derivation of the absorption spectra..28 .˘ Derivative Spectrophotometric Determination of. G.4 190 200 20 0 -20 -40 -62. together with the spectra of standard caﬀeine solutions prepared in water.23 (a) (b) 93..35 340 350 220 240 260 280 nm 300 320 Figure 2. (c) tea (—-). This eﬀect is clearly seen in Fig. The concentration of caﬀeine in the sample solutions was deduced by means of the regression equation of the related calibration graph. which shows the absorption spectra of cola. some tedious background correction techniques have been used27. ALPDOGAN.66 80 60 40 1D 20 1D 20 1D 1D 20 0 -20 -20 -20 -40 -43. the derivatization of the absorption spectrum and measurement of the distance between two neighboring extremum values allow the elimination of matrix eﬀects.

01 (r = 0.42 2 7.79 340 350 220 240 260 280 nm 300 320 -6.071c + 0. For the comparison of the derivative spectrophotometric method with HPLC samples were also analyzed on a C 18 reversed-phase column using MeOH-H2 O (30:70 v/v) mobile phase.86 320 340 350 −1 0 0 2D 2D 2D -2 -2 -4 -4 -2 -4 -6 -7.0731c + 0.79 190 200 -6 -7.-) solutions. Concentration: 6 µg ml−1 .. In the chromatogram obtained by this procedure.56 4 2 0 2D -2 -4 -6 -8 -9..4 190 200 6.0-10.86 190200 220 240 260 280 nm Figure 3. there was no signiﬁcant diﬀerence between the mean values and precisions of the two methods.9999) (Tea) (D3 ) = 0. As shown in Table 1. having a retention time of 3.56 (c) 4 2 0 -2 -4 -6 -8 2D 300 320 -6.35 6 4 2 0 -2 -4 2D 220 240 260 280 nm 6.. G.01 (r = 0.4 340 350 300 -9. the retention time of caﬀeine is 5..˘ Derivative Spectrophotometric Determination of.42 (a) 2 3. Reference: Water Linear relationships were obtained between the caﬀeine concentration (2. and caﬀeine (.. Second order derivative spectra of (a) cola (—-). (c) tea (—-).14 min. acetaminophen was used. The regression equations were as follows: (D3 ) = 0.03 min. As the internal standard substance.03 (r = 0.35 6 4 2 0 (b) 7. 3.25 c + 0.9999) (Coﬀee) (c: µg ml (D3 ) = 0.0 µg ml−1) and peakto-peak amplitudes (D 3 ) of third order spectra and (D 2 ) of second order spectra at the wavelengths stated above. The assay results obtained by both methods were statistically compared at 95% conﬁdence level. (b) coﬀee (—-).9998) (Cola) The contents of caﬀeine in six samples were determined by the proposed method. et al. 299 ) . ALPDOGAN..

Comparison of the results for caﬀeine determination by the proposed method and the HPLC method Derivative Spectrophotometric Method Methods HPLC Method 150.5 -1.0 -2.5 3D -1..25 1.38 1. (b) coﬀee (—-).5 -1.0 3D 3D -0.5 3D 1.5 0.53 ± 0. coﬀee and tea...0 -1.36 ± 0.03 1. and caﬀeine (.5 -1.5 -1.05 for p = 0.0 -1.05 1. ALPDOGAN.5 0.04 t-test of signiﬁcance* 0.25 340 350 -1.43 340 350 -2.5 -1.43 1.0 -1.05 and n1 = n2 = 6 √ Mean ± t. Third order derivative spectra of (a) cola (—-).23.0 -0.5 -1. the derivative spectrophotometric method is relatively easy. Reference: Water Table 1.0 0.5 (a) 0.51 ± 0.0 -2. Because it does not require expensive solvents and reagents.5 (b) 0.5 0.0 (c) 0. it may be recommended for the rapid.0 0.0 -0.75 0.00 In conclusion.5 -2.-) solutions. et al.32 ± 0.68 1. fast and cheap for the determination of the caﬀeine content of cola.71 0.0 -0.0 -0.0 -1.5 0. (c) tea (—-).70 4.5 -2.0 -1.0 -2. precise and sensitive quantiﬁcation of caﬀeine in these 300 3D .73 190 200 3D 220 240 260 280 nm 300 320 Figure 4.45 F-test of signiﬁcance* 4.73 340 350 260 280 nm 300 320 1.06 ± 1.43 1.03 1.05 0.0 -0.5 0.0 -2.... 0.75 0.43 190 200 220 240 260 280 nm 300 320 0.78 *t = 2. G.25 190 200 -2.35 ± 0.25 220 240 1.s / n Cola Coﬀee Tea (%) (%) (µg ml−1) 149. F = 5.˘ Derivative Spectrophotometric Determination of.5 -1. Concentration: 6 µg ml −1 .

. Y. Mikhailova. The major instrument required is a modern.A. J. 73-76 (1985). 84932 z ( 1967 ) 7.. Shokuhin Eiseigaku Zashi. Rockville MD. 143 (4). Hungnan. Monokova. 85. 22375 t (1989) 18. Ref. K. ALPDOGAN. Maede. Delaney. Tencheva. Ref.. Ref. C. Ref. 78.. P. O. 108. C. Method detection limits (MDLs) were 2 µg/g. 10111 m (1973). 14. S. 129. Chromatographia.C. 741 ( 1967 ). 3.. Riederer P. S. Lebensmittelunters.Ref. Duijn. Sci. 67. Ref. 61.A. 52640 w (1963). 92. G. 561-565 (1988).Y. Kunugi. Korologas . Ref. Hollis. Ref. Lett. Yakugaka Zassbi. Educ. Prog. Kuit. The United States Pharmacopeia.. Washington. A. 9621 d (1965) 6. 5. 43452 r (1980). J. 169060 z (1988). 141656 v (1989) 11.. Kurzidin. Int..A. 106. M. Y. 83 (1965). Anal. 107.Khayyal. et al. K. 46(10). 152985 r ( 1987 ). D. Oi Inoba.A. London Her Majesty’s Stationery Oﬃce Vol. 62 (7).A. Laskowsi.Chem. Gangye. ( 1984 ). This method also takes less time than the others. Forsch. 110. 13. C. 206633 r (1987).A. A. Sasaki. 11th 227-244 (1985). P. L.. Chakurova. 73903 e (1988). 9(5).. V. United States Pharmacopeial Convention. 87(6). 460 (1976). Anal.˘ Derivative Spectrophotometric Determination of..P. S. Zhardaniye. Chromatog. D. E.. Colloq. 76415 k (1977) 8. Chen Jiahua.A.Krueger. M. R. G. Linda. J.I 1993. Kathleen. respectively. 15. 107-115 (1988).Chromatogr. 199 (1979) 21. H.. Z.. Krolikovski. microcomputerized UV/VIS spectrophotometer.. 2 µg/g of caﬀeine for tea.A. Kazhi. 17.5 µg ml−1 . 16.. 35. Banchher E. British Pharmacopeia.. (1). Von.A. 618-620 (1985)..A. Ref. (1990) 2. Vander Stegen. 86. T.. 211 (1976) Ref. and the absorption values of the solutions are stable for at least 2 days. U. Y. Probl. Z. H.. P. Sepu. P.. A.. Dunkckova. Ref. Revision.C. C. Farm. C. Mueller.. T.. Aoki.A. (1). Farmakol. cola and coﬀee. Ref.. 29 (2). Przem. 4.. Farm. References 1. 253 (1973). 179 (1). C. 43-48 (1987).Hoeﬄer.Savior.. 106.. Oﬃcial Methods of Analysis.A. C.. which can be purchased at a reasonable price. M. 48717 y (1987) 19. Z. J. 16(B19). Kunogi. AOAC Arlington VA. C. 99 (1979)..Shengging. J. 9(1). 86823 k (1976) 9.A. 1. E. N. Razniki Inst. J. 367-369 (1987). 301 . products.Ayad. J. 1682 (1963). 109. 110. C. C. Flovur Res. The method can be extended to caﬀeine determination in various beverages.F. Masatauch. Humprey. 10. Yamazue. 22nd . Cafe. Subtrop. 2. 441 (1977). Ref. Bunseki Kagaku 37 (11). H. Coggon. J. Kitada. 5(6). Dzneladze. 136-140 (1988). C. Washuetti. Miec. 12. 20. C.C. 65. Obz.A. provided that the proper wavelengths for peak-to-peak measurements are accurately selected.F. 1525 (1983). S. Inc. Chem. Ref.

.K. An. Bai.J. 30. Abstr. 98 (1988) Ref. A. 51 Anal. C.F. 50 (1988) 8 F 51. Luk. lll. Forsch. Acar. Y.S. 51 (1989) 2 F 46. Majun. Lebensm . Abdel-Moety. N.. C. L. Die Pharmazie . 616 (1988). O.109. Xu. S.Unters.. Dai. 885 (1999) 27. et al. U. L. 105.˘ Derivative Spectrophotometric Determination of. Zangjie. 28. S. Wong. Ref. 22. Randi. 23 (8). Yauxue Xuebau. Ref. Ref. G. Jia. P. 110. Lu. 825991 (1989) ¨ 25. Sungur. Ersoy. 5 (2). S. Fung. Analyst.M. 441 (1988). Shipin Kexue (Beijing). Deng. Yauxue Xuebau. 23 (6). T. Li. A.. Z. Luk. H. 90 (1986 ). Yurdakul. . Onur. 29.. Anal. 665 (1986). A.S. K. Gazi Univ. C. 186 (1988) Ref. Abstr. S. C. S. J.F. Hongwei. 46. Analyst. Y. Jiaku. G. Yauxueyuan Xuebau 3 (2).W. Lau. F. . 110. E. S. 412. A. . 135053 j (1988) 24.S. 214148 q (1986) 23. P. 302 . S. Fac. 167 (1988) 26.. Dang.H. 201 (1985). ALPDOGAN.

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