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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

. Experimental Apparatus For derivative spectrophotometric measurement a Philips 8740 UV-VIS spectrophotometer and 10 mm quartz cells were used. Internal standard (acetaminophen) solution (100 µg ml−1 ) was prepared. Quantitative analysis was accomplished by internal standard calibration.0 g) was homogenized with 100 ml of water using a magnetic stirrer for about 5 min. 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. 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.0 µg ml−1). The ﬂow rate was 1.˘ Derivative Spectrophotometric Determination of.5 ml min−1 (pSI=2600). The mixture was cooled. Chromatographic analyses were performed using a Waters HPLC system consisting of a model 510 delivery system. Materials Caﬀeine and acetaminophen (internal standard) (Do˘ gu) were used as received. In order to degas the cola.The portions of each sample were analyzed using two methods. The ﬁrst 20 ml was discarded and the ﬁltrate was used for the analysis. ALPDOGAN. method27.. et al. Several cola. 296 . coﬀee and tea were chosen owing to their high caﬀeine content. This solution was freshly prepared and was diluted to obtain standard solutions for the preparation of calibration curves. Some visible spectrophotometric methods often require tedious pre-separation techniques to remove possible interferences from colored materials.. 500 nm min−1 scan speed and very high smoothing. A 10 µm Bondapak C18 (3. Milli Q water was used. Merck. All spectra were recorded from 190 nm to 350 nm with 2 nm slit width. HPLC grade methanol and other analytical grade chemicals were purchased from E. coﬀee and tea samples were analyzed for their caﬀeine content and the resuts were compared with those obtained by the HPLC method.5-2. The samples were then diluted to provide the necessary working concentration (2. and for eliminating the eﬀect of baseline shifts and baseline tilts30 . G. the sample solution was mixed for 20 min by with a magnetic stirrer. Caﬀeine stock solution (100 µg ml−1) was prepared by dissolving 10 mg of caﬀeine in 100 ml of water. Derivative spectrophotometry is now a reasonably priced standard feature of modern microcomputerized UV/Vis spectrophotometry.28 . 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. Analytical Procedures Preparation of sample solution Samples were purchased from the local markets for analysis . Derivative spectrophotometry is a useful technique for extracting qualitative and quantitative information from spectra composed of unresolved bands29 . Soluble coﬀee (1.9 mm x 30 cm) RP column and an isocratic mobile phase containing MeOH-water (30:70) were used.0-10.. Cola.

0 .1 nm were used for the quantitative determination of caﬀeine in cola. ALPDOGAN.0 190 200 220 240 260 280 nm 300 320 0. (c) tea (—-). Reference: Water Sample Analysis Second and third order derivative spectra of sample solutions were recorded against water.0 ABS 1.5 0.5 1.0 2.5 1... and caﬀeine (.0 1.0 2.5 2.0 2.0-300.0 0. G. In the third order derivative spectrum..3 nm were used for tea.5 2.5 1.0 (b) 3.0 -10.0 3.5 2. the extrema of 246.˘ Derivative Spectrophotometric Determination of.2 nm in the second derivative spectrum for cola. the extrema of 286.5 1.0 2..5 0.5 nm in the third derivative spectrum for coﬀee.0 1.0 190 200 220 240 260 280 nm 300 320 0.0 2. 268.0 1.0 nm in the third derivative spectrum for tea samples.7 nm and 234.0 2.5-289. Regression equations of the calibration graphs were calculated by the method of least squares.0 0.0 (a) 3.7-245. (b) coﬀee (—-). and the extrema of 286. Peak-to-peak measurements were made at 232. Preparation of Calibration Graphs The second and third order derivative absorption spectra of caﬀeine standard solutions (2.5 1. Concentration: 6 µg ml−1 .5 0.7 nm and 300. In the second order derivative spectrum..5 0. and 286.0 340 350 3. 297 ABS 2.. Absorption spectra of (a) cola (—-).5 0.5 0.5 ABS 2. 3.0 190 200 220 240 260 280 nm 300 320 0.0 340 350 Figure 1.0 (c) 3.0 340 350 0.7 nm and 265 nm were used for coﬀee.5 1.-) solutions. et al.0 µg ml−1) were recorded against water between 190 and 350 nm.5 2.0 1.5 ABS ABS ABS 1.

. G. coﬀee and tea extract. First order derivative spectra of (a) cola (—-). As seen. 93. In contrast. Reference: Water 298 1D .23 (a) (b) 93. second and third order derivatives of the absorption spectra given in Fig. and caﬀeine (. 1.4 190 200 20 0 -20 -40 -62.19 190 200 220 240 260 280 nm 300 320 -43. although extremum wavelengths are not exactly same as those obtained with standard caﬀeine solutions.35 340 350 220 240 260 280 nm 300 320 Figure 2.. 3 and 4 show ﬁrst. because the variable background absorptions overlapping the analyte peaks are smoother in derivative spectra. the derivatization of the absorption spectrum and measurement of the distance between two neighboring extremum values allow the elimination of matrix eﬀects. (c) tea (—-).28 . ALPDOGAN.03 60 40 20 0 -20 -40 -60... Figs. together with the spectra of standard caﬀeine solutions prepared in water.. et al. (b) coﬀee (—-).4 340 350 68. some tedious background correction techniques have been used27.23 (a) 40 68.. 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.˘ Derivative Spectrophotometric Determination of. The concentration of caﬀeine in the sample solutions was deduced by means of the regression equation of the related calibration graph. 1.19 340 350 -62.66 80 60 40 40 0 0 220 240 260 280 nm 300 320 78.03 60 (c) 40 1D 20 0 -20 -40 -60. To overcome this diﬃculty. which shows the absorption spectra of cola.35 190 200 78.66 80 60 40 1D 20 1D 20 1D 1D 20 0 -20 -20 -20 -40 -43. matrix eﬀects are eliminated by derivation of the absorption spectra.-) solutions. This eﬀect is clearly seen in Fig. 2. Concentration: 6 µg ml−1 .

25 c + 0.03 min. having a retention time of 3.42 2 7. 299 ) . G. (b) coﬀee (—-). In the chromatogram obtained by this procedure.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. and caﬀeine (.01 (r = 0.. As shown in Table 1. the retention time of caﬀeine is 5.03 (r = 0..0731c + 0.35 6 4 2 0 -2 -4 2D 220 240 260 280 nm 6.9999) (Tea) (D3 ) = 0.4 190 200 6. 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. As the internal standard substance.79 190 200 -6 -7. et al. there was no signiﬁcant diﬀerence between the mean values and precisions of the two methods.˘ Derivative Spectrophotometric Determination of... The assay results obtained by both methods were statistically compared at 95% conﬁdence level. acetaminophen was used. (c) tea (—-).-) solutions. Second order derivative spectra of (a) cola (—-).14 min. Reference: Water Linear relationships were obtained between the caﬀeine concentration (2. The regression equations were as follows: (D3 ) = 0. Concentration: 6 µg ml−1 .. ALPDOGAN.79 340 350 220 240 260 280 nm 300 320 -6.9998) (Cola) The contents of caﬀeine in six samples were determined by the proposed method.9999) (Coﬀee) (c: µg ml (D3 ) = 0. 3.86 190200 220 240 260 280 nm Figure 3.35 6 4 2 0 (b) 7.56 4 2 0 2D -2 -4 -6 -8 -9.071c + 0.0-10.01 (r = 0.42 (a) 2 3.4 340 350 300 -9.56 (c) 4 2 0 -2 -4 -6 -8 2D 300 320 -6.

0 -2.35 ± 0.0 -0.0 -0. the derivative spectrophotometric method is relatively easy. G.75 0. precise and sensitive quantiﬁcation of caﬀeine in these 300 3D .05 1.71 0. et al. ALPDOGAN.s / n Cola Coﬀee Tea (%) (%) (µg ml−1) 149.5 0.0 -1.45 F-test of signiﬁcance* 4.43 1.03 1.5 -1.0 0.0 -1.0 -2.5 0. Because it does not require expensive solvents and reagents.00 In conclusion.23.0 -1.05 0. it may be recommended for the rapid.43 340 350 -2.. fast and cheap for the determination of the caﬀeine content of cola.05 for p = 0.5 -1.06 ± 1.5 -1. Reference: Water Table 1.05 and n1 = n2 = 6 √ Mean ± t.0 -0.78 *t = 2.03 1.. and caﬀeine (.5 0..5 0.0 -1.25 220 240 1.04 t-test of signiﬁcance* 0.0 -2.0 -0. (c) tea (—-).. 0.0 (c) 0.68 1. Third order derivative spectra of (a) cola (—-).˘ Derivative Spectrophotometric Determination of.73 340 350 260 280 nm 300 320 1.5 (a) 0.53 ± 0.38 1.5 -2. coﬀee and tea.0 -2.-) solutions.5 (b) 0.0 3D 3D -0.36 ± 0.32 ± 0.43 190 200 220 240 260 280 nm 300 320 0.0 -1.43 1.5 -1.0 -0.0 0.70 4.5 -1. Concentration: 6 µg ml −1 . F = 5.5 3D 1.5 0.51 ± 0.5 -1.25 1.25 190 200 -2.25 340 350 -1.75 0. Comparison of the results for caﬀeine determination by the proposed method and the HPLC method Derivative Spectrophotometric Method Methods HPLC Method 150.5 3D -1..5 -1. (b) coﬀee (—-).5 -2.73 190 200 3D 220 240 260 280 nm 300 320 Figure 4..

Ref. 211 (1976) Ref. Anal. 169060 z (1988). J. Linda. Kazhi. Farm. Coggon. 29 (2). 48717 y (1987) 19. M. H. Sasaki. Probl. 2. 52640 w (1963). J.P. R. References 1. K. This method also takes less time than the others. 109. Flovur Res.Ref. Kitada.. C. The major instrument required is a modern. 85.. N. C. 43452 r (1980).A.Shengging. O. Lebensmittelunters. Ref. 106. Y. 83 (1965).Hoeﬄer.A. Chakurova. Int.. Sepu. Farm. Z.. 73-76 (1985). Kathleen. 179 (1). A. P. 9621 d (1965) 6. S. Korologas . T.A. The United States Pharmacopeia. Cafe. Lett. 22nd .Khayyal. C. Chromatog.Krueger. T. Ref.Chromatogr. Krolikovski. Masatauch. 460 (1976).. J. United States Pharmacopeial Convention. P. Y. respectively. et al. Mueller. Ref..Chem. Miec. 110. Washuetti. 62 (7). Chem. 741 ( 1967 ). Delaney. 107. 13.. Ref. 14. Duijn.. ( 1984 ). Gangye. 15.A. (1). London Her Majesty’s Stationery Oﬃce Vol. Inc. Chromatographia. 16(B19). 87(6). E. U.. Yakugaka Zassbi. Maede. (1). 253 (1973). Kunogi.A. Bunseki Kagaku 37 (11). Rockville MD. Monokova. 99 (1979). J. C. 143 (4). 9(5). P.A. Ref. 20. D. 86.. 22375 t (1989) 18. 10111 m (1973). G. 35. products. Ref. 3. 1525 (1983). J. D. Ref. 11th 227-244 (1985). 16. Shokuhin Eiseigaku Zashi.A. C.A. 46(10). E. Przem..5 µg ml−1 . S. Banchher E. Subtrop. S. microcomputerized UV/VIS spectrophotometer. 108. which can be purchased at a reasonable price. (1990) 2. 2 µg/g of caﬀeine for tea. provided that the proper wavelengths for peak-to-peak measurements are accurately selected. P. Tencheva. 129. 561-565 (1988). 106. Ref. J. 367-369 (1987). Forsch. 152985 r ( 1987 ). Anal. C. 17. C. and the absorption values of the solutions are stable for at least 2 days. J. G.Y. A. 4. Hungnan. 43-48 (1987). Prog. 5. Educ. H.A. 78. Z. 73903 e (1988)... Kurzidin. M. 84932 z ( 1967 ) 7. H.C. Ref. 9(1). Dunkckova. 136-140 (1988). British Pharmacopeia. Y.. Ref. S. C. Humprey. Farmakol.Savior.A. 61. K. Yamazue. AOAC Arlington VA...F. Washington. Aoki. 199 (1979) 21. Riederer P. Ref..I 1993. Obz. Colloq. C. Oi Inoba. ALPDOGAN.A.C... Sci. 86823 k (1976) 9.A. Zhardaniye. 107-115 (1988). Laskowsi. C. Vander Stegen.... 67. Oﬃcial Methods of Analysis.. 1682 (1963). 5(6).F. 110. cola and coﬀee. Dzneladze. 1. Kunugi. Razniki Inst. Chen Jiahua.A. Z. Method detection limits (MDLs) were 2 µg/g.Ayad. 76415 k (1977) 8. 92.˘ Derivative Spectrophotometric Determination of.. L. Revision.. Kuit. Von. Mikhailova. 10.A.. V. C.. 618-620 (1985). 206633 r (1987). J. M.. 65. 12.. 301 . A. Hollis. C. The method can be extended to caﬀeine determination in various beverages.. 441 (1977). 141656 v (1989) 11.C.

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

determinación espectrofotometrica de la cafeina

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