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¨ ˙ c TUBITAK

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

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

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

296 . the sample solution was mixed for 20 min by with a magnetic stirrer. Derivative spectrophotometry is now a reasonably priced standard feature of modern microcomputerized UV/Vis spectrophotometry. Quantitative analysis was accomplished by internal standard calibration. ALPDOGAN. Soluble coﬀee (1. The ﬂow rate was 1. 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. a model 481 variable wavelength spectrophotometer (at 270 nm) and a computer.˘ Derivative Spectrophotometric Determination of. Milli Q water was used. In order to degas the cola. Cola. The ﬁrst 20 ml was discarded and the ﬁltrate was used for the analysis. Analytical Procedures Preparation of sample solution Samples were purchased from the local markets for analysis .0 g) was homogenized with 100 ml of water using a magnetic stirrer for about 5 min. Experimental Apparatus For derivative spectrophotometric measurement a Philips 8740 UV-VIS spectrophotometer and 10 mm quartz cells were used. This solution was freshly prepared and was diluted to obtain standard solutions for the preparation of calibration curves.9 mm x 30 cm) RP column and an isocratic mobile phase containing MeOH-water (30:70) were used. and for eliminating the eﬀect of baseline shifts and baseline tilts30 .5-2. Internal standard (acetaminophen) solution (100 µg ml−1 ) was prepared. A 10 µm Bondapak C18 (3.0 µg ml−1).5 ml min−1 (pSI=2600). Materials Caﬀeine and acetaminophen (internal standard) (Do˘u) were used as received.The portions of each sample were analyzed using two methods. coﬀee and tea samples were analyzed for their caﬀeine content and the resuts were compared with those obtained by the HPLC method. Several cola. method27. All spectra were recorded from 190 nm to 350 nm with 2 nm slit width. Merck. et al.. coﬀee and tea were chosen owing to their high caﬀeine content. G. Chromatographic analyses were performed using a Waters HPLC system consisting of a model 510 delivery system.... The mixture was cooled. 500 nm min−1 scan speed and very high smoothing. Some visible spectrophotometric methods often require tedious pre-separation techniques to remove possible interferences from colored materials.0-10. The samples were then diluted to provide the necessary working concentration (2.28 . 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. Derivative spectrophotometry is a useful technique for extracting qualitative and quantitative information from spectra composed of unresolved bands29 . HPLC grade methanol and g other analytical grade chemicals were purchased from E. diluted to 100 ml with water and ﬁltered through a dry ﬁlter paper. Caﬀeine stock solution (100 µg ml−1) was prepared by dissolving 10 mg of caﬀeine in 100 ml of water.

.5 2.0 (a) 3. 297 ABS 2.0 0. 3.0 1.5 2. (c) tea (—-).0 -10.5 0..5 2.5 1. et al.0 190 200 220 240 260 280 nm 300 320 0.0 1. In the third order derivative spectrum.0 (c) 3.0 (b) 3.5 0.7 nm and 265 nm were used for coﬀee. (b) coﬀee (—-).0 µg ml−1) were recorded against water between 190 and 350 nm.0-300.-) solutions. 268.0 2. and the extrema of 286.0 nm in the third derivative spectrum for tea samples.3 nm were used for tea. Concentration: 6 µg ml−1 .0 ..5 1.5 ABS ABS ABS 1.0 2.0 190 200 220 240 260 280 nm 300 320 0. Absorption spectra of (a) cola (—-).0 340 350 3.5 ABS 2.5 1. the extrema of 286.5 nm in the third derivative spectrum for coﬀee..0 340 350 0.0 ABS 1.0 2. the extrema of 246. G.0 340 350 Figure 1.1 nm were used for the quantitative determination of caﬀeine in cola. ALPDOGAN.˘ Derivative Spectrophotometric Determination of.0 2.5 0. Peak-to-peak measurements were made at 232.0 2.5 1.7 nm and 300. Preparation of Calibration Graphs The second and third order derivative absorption spectra of caﬀeine standard solutions (2.0 1.7-245.5 0.5 1.5 2. Regression equations of the calibration graphs were calculated by the method of least squares. and 286.2 nm in the second derivative spectrum for cola. In the second order derivative spectrum.0 0.0 1..5 0.0 190 200 220 240 260 280 nm 300 320 0.5 1.0 3.. and caﬀeine (.7 nm and 234. Reference: Water Sample Analysis Second and third order derivative spectra of sample solutions were recorded against water.5-289.0 2.5 0.

Figs.28 .66 80 60 40 1D 20 1D 20 1D 1D 20 0 -20 -20 -20 -40 -43. some tedious background correction techniques have been used27. because the variable background absorptions overlapping the analyte peaks are smoother in derivative spectra. 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.19 190 200 220 240 260 280 nm 300 320 -43. This eﬀect is clearly seen in Fig.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.˘ Derivative Spectrophotometric Determination of. et al. together with the spectra of standard caﬀeine solutions prepared in water.. Reference: Water 298 1D .4 190 200 20 0 -20 -40 -62. which shows the absorption spectra of cola. In contrast..35 190 200 78. (b) coﬀee (—-). 2. Concentration: 6 µg ml−1 .03 60 40 20 0 -20 -40 -60. As seen. ALPDOGAN.4 340 350 68.23 (a) 40 68. To overcome this diﬃculty. matrix eﬀects are eliminated by derivation of the absorption spectra. the derivatization of the absorption spectrum and measurement of the distance between two neighboring extremum values allow the elimination of matrix eﬀects. 3 and 4 show ﬁrst. G. The concentration of caﬀeine in the sample solutions was deduced by means of the regression equation of the related calibration graph.19 340 350 -62. First order derivative spectra of (a) cola (—-). and caﬀeine (.-) solutions.23 (a) (b) 93. coﬀee and tea extract. 1.35 340 350 220 240 260 280 nm 300 320 Figure 2. (c) tea (—-). although extremum wavelengths are not exactly same as those obtained with standard caﬀeine solutions.. 93. second and third order derivatives of the absorption spectra given in Fig... 1.

86 320 340 350 −1 0 0 2D 2D 2D -2 -2 -4 -4 -2 -4 -6 -7. The regression equations were as follows: (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.. In the chromatogram obtained by this procedure.86 190200 220 240 260 280 nm Figure 3. 299 ) .9999) (Coﬀee) (c: µg ml (D3 ) = 0.9999) (Tea) (D3 ) = 0. Second order derivative spectra of (a) cola (—-)..56 (c) 4 2 0 -2 -4 -6 -8 2D 300 320 -6. As shown in Table 1.01 (r = 0. acetaminophen was used. there was no signiﬁcant diﬀerence between the mean values and precisions of the two methods.. the retention time of caﬀeine is 5. 3. Reference: Water Linear relationships were obtained between the caﬀeine concentration (2.0-10.56 4 2 0 2D -2 -4 -6 -8 -9. Concentration: 6 µg ml−1 .42 2 7.9998) (Cola) The contents of caﬀeine in six samples were determined by the proposed method. and caﬀeine (. ALPDOGAN.˘ Derivative Spectrophotometric Determination of.79 190 200 -6 -7.42 (a) 2 3.4 340 350 300 -9.35 6 4 2 0 (b) 7.071c + 0.25 c + 0.. et al. As the internal standard substance.01 (r = 0.. having a retention time of 3.4 190 200 6. The assay results obtained by both methods were statistically compared at 95% conﬁdence level.35 6 4 2 0 -2 -4 2D 220 240 260 280 nm 6.79 340 350 220 240 260 280 nm 300 320 -6.03 (r = 0.0731c + 0. (c) tea (—-).-) solutions..03 min. (b) coﬀee (—-). G.14 min. 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.

0 -2. Comparison of the results for caﬀeine determination by the proposed method and the HPLC method Derivative Spectrophotometric Method Methods HPLC Method 150.32 ± 0.43 1.06 ± 1.0 3D 3D -0.25 340 350 -1.5 -1.03 1.5 -1.0 -2.5 0.0 -0.0 -2.0 -2. 0.03 1.05 for p = 0.05 and n1 = n2 = 6 √ Mean ± t.78 *t = 2.0 (c) 0.0 -1. (b) coﬀee (—-).5 (a) 0.5 (b) 0.36 ± 0. and caﬀeine (.25 190 200 -2.5 3D 1.23.53 ± 0. Third order derivative spectra of (a) cola (—-).73 190 200 3D 220 240 260 280 nm 300 320 Figure 4.00 In conclusion..45 F-test of signiﬁcance* 4.0 -0. Because it does not require expensive solvents and reagents.0 0.35 ± 0. Reference: Water Table 1.04 t-test of signiﬁcance* 0.5 0.5 -1.43 1.75 0. the derivative spectrophotometric method is relatively easy.68 1.0 -1.38 1.43 340 350 -2.5 -1.05 0. (c) tea (—-). ALPDOGAN.0 -0.25 220 240 1.71 0.5 -1.5 0.0 -1.43 190 200 220 240 260 280 nm 300 320 0. F = 5. precise and sensitive quantiﬁcation of caﬀeine in these 300 3D . it may be recommended for the rapid.-) solutions.51 ± 0.. coﬀee and tea.5 -1.s / n Cola Coﬀee Tea (%) (%) (µg ml−1) 149. G.5 0.0 0.. et al.0 -1.70 4. fast and cheap for the determination of the caﬀeine content of cola. Concentration: 6 µg ml −1 .5 -2.05 1..5 -1.75 0.0 -0.0 -0.5 0.73 340 350 260 280 nm 300 320 1.5 -2.5 3D -1..˘ Derivative Spectrophotometric Determination of..0 -1.25 1.

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

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

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