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    Determination and Characterization of Caffeine in Tea, Coffee and Soft Drinks by Solid Phase Extraction and High Performance Liquid Chromatography (SPE – HPLC) Md. Abdul Mumin, Kazi Farida Akhter, Md. Zainal Abedin*, Md. Zakir Hossain. Department of Chemical Engineering and Polymer Science, Shah Jalal University of Science and Technology, Sylhet-3114, Bangladesh. *Correspondence should be addressed: abedin@sust.edu.  Abstract : Caffeine (1,3,5-trimethylxanthine), a mild addicting drug was isolated, purified and characterized from tea (black and green) and coffee. Isolation was done by liquid-liquid extraction using chloroform as an extracting solvent. Extraction was carried out in four steps such as leaching, dye removal, liquid extraction and recrystallization. Crude caffeine was purified by solid phase extraction (SPE) method. The solvent used for recrystallization were toluene and petroleum ether. The purity of caffeine was ascertained by the determination of melting temperature. Pure caffeine obtained from different samples was characterized by UV-Visible spectrophotometer, TLC, FT-IR and HPLC. In HPLC, 50mM KH 2 PO 4  (pH=2), acetonitrile and methanol (40 : 8 : 2) was used as solvent as well as mobile phase. Amount of caffeine in various soft drinks (Cola) commercially available in Bangladesh was also determined by HPLC method. Key Words: ã Caffeine, ã Chloroform, ã SPE, ã HPLC, ã KH 2 PO 4 , ã Acetonitrile. Received : 15.02.06; accepted : 9.11.06  Introduction Caffeine is a naturally occurring substance found in the leaves, seeds or fruits of over 63 plants species worldwide and is part of a group of compounds known as methylxanthines. The most commonly known sources of caffeine are coffee, cocoa beans, cola nuts and tea leaves. Caffeine is a pharmacologically active substance and depending on the dose, can be a mild central nervous system stimulant. Caffeine does not accumulate in the body over the course of time and is normally excreted within several hours of consumption [1]. Caffeine is an alkaloid of the methylxanthine family. In its pure state, it is an intensely bitter white powder. Its chemical formula is C 8 H 10 N 4 O 2 , its systematic name is 1,3,5-trimethylxanthine [2] and its chemical formula is shown below. Structure of caffeine Pure caffeine occurs as odorless, white, fleecy masses, glistening needles of powder. Its molecular weight is 194.19g, melting point is 236 0 C, point at which caffeine sublimes is 178 0 C at atmospheric pressure, pH is 6.9 (1% solution), specific gravity is 1.2, volatility is 0.5%, vapor pressure is 760 mm Hg at 178 0 C, solubility in water is 2.17%, vapor density 6.7 [3] Decaffeination is a popular term in present modern world to optimize the caffeine contents in various sources. This is simply use of a solvent, which extract caffeine. For this purpose, the currently available solvents are chloroform, methyl chloride, ethyl acetate, super critical carbon dioxide etc. Methylene chloride is used to decaffeinate a high proportion of conventional teas. As a solvent, methylene chloride is highly effective, but also potentially dangerous under certain circumstances. It can cause faintness, dizziness, and headache if inhaled at high concentrations. Ethyl acetate is another compound used to extract caffeine from tea. It removes caffeine from tealeaves effectively; it can also extract other chemical components as well. Studies on green tea decaffeinated with ethyl acetate have shown the potential for up to 30% of epigallocathechin gallate (EGCG-considered to be the primary beneficial component in green tea) and other beneficial antioxidant compounds to be extracted along with the caffeine [4-6]. Ethyl acetate is also moderately toxic.  Malaysian Journal of Chemistry, 2006, Vol. 8, No. 1, 045 - 051      Super critical carbon dioxide (CO 2 ) is the fancy name for using highly pressurized carbon dioxide—the gas that adds bubbles to mineral water—to dissolve caffeine from tealeaves. The advantages of CO 2  are that it does not leave a chemical residue and it has a minimal effect on the flavor and beneficial compounds inherent to the tea [6]. Though chloroform is toxic, in laboratory purposes it is best caffeine extracting solvent and the results obtained by characterization becomes satisfactory and reproducible always [7]. In the present study, we have extracted caffeine from green tea, black tea, coffee and then characterized by melting point,   max (UV- Visible), IR absorption bands, R f   (TLC) and RT (HPLC). One of the major objectives is to develop an improved purification method based on Solid phase extraction. We have also developed an easily adaptable HPLC method for both qualitative and quantitative determination of Caffeine. In Bangladesh, there is no authentic data about caffeine content in soft drinks. So, we also determined the concentration of caffeine in various available soft drinks, especially cola drinks. The proposed method is simple, rapid and has significant advantages over spectrophotometric methods as well as other HPLC methods. Experimental  Materials: KH 2 PO 4 (Merck, Germany); Phosphoric acid (85 %, Merck); Petroleum ether (BDH, England); Standard Caffeine (Merck, Germany); Lead Ethnoate, Chloroform, Toluene, Methanol, Acetonitrile(Merck, Germany). Black tea (Ispahani Mirzapur tea), Coffee (Nescafe) and various brand soft drinks were collected from local market and green tea from Bangladesh Tea Research Institute (BTRI).  Apparatus: HPLC; Model: Waters 515, with UV Detector & Vacuum filter, pH   Meter (REX, Model p HS-25 ), Visi TM -1 SPE Single Sample Processor (Supelco) and UV-Visible spectrophotometer (UV-1601,Shimadzu, Japan), Melting Electrical melting point apparatus (Electrothermal, IA9100), FT-IR (SHIMADZU, Model-IR Prestige-21), TLC (Whatman, 250  m layer, 20X20 cm). Methods  Isolation of caffeine from tea and coffee:   5 gm solid tea was taken in 500 ml beaker and subsequently distilled water (225 ml) was added to it. The mixture was boiled for 10 min and filtered by using a Buchner Funnel. 10% lead ethnoate solution (25ml) was added with the filtrate, boiled for 5 min and filtered again. The purpose of the addition of 10% lead ethnoate is to convert any extracted tannins or other acids into anions. As electrically neutral polar molecules, the acids tend to be soluble in both water and CHCl 3 , complicating the purification and increasing the tendency to form an emulsion in the next step. As anions they are not soluble in CHCl 3 and this helps to avoid an emulsion. The 10% lead ethnoate seems to cause precipitation of some substances that tend to clog the filter paper in the next step and also helps avoid formation of emulsions. A 500 ml separatory funnel was put into an iron ring on a ring stand. Pouring the tea solution in the separatory funnel and adding about 30 ml of CHCl 3 , the solution was shaken uniformly while stopcock was opened to expel vapors. The layers were allowed to separate and the lower layer (chloroform) was collected into a 100ml beaker and the separation procedure was repeated for the second time to collect into the beaker. Anhydrous sodium sulfate was added in the beaker containing the combined extracts. The anhydrous sodium sulfate would act to remove any water and water-soluble salts that were retained in the chloroform (organic layer) or accidentally transferred during decantation. The beaker containing the extract was then heated a short period for dryness using a water bath and the temperature was controlled low enough at 70-90 0 C to avoid caffeine decomposition. After 24 hours, white crude caffeine obtained at the bottom of the beaker. The crude caffeine obtained from the above method was purified and recrystallized by solid phase extraction method using chloroform, toluene and petroleum ether. In case of coffee, 1 gm coffee was taken into a 500 mL beaker and subsequently 250mL hot distilled water was added. Coffee was properly dissolved in the hot distilled water. Then the whole procedure was same as that of isolation of caffeine from tea. Solid Phase Extraction (SPE): Commercial tea and coffee consists of many components that cause chromatographic interferences with caffeine. For this reason the sample treatment proposed consists of SPE with Sep-Pak C 18 (500 mg) cartridges that enable separation of caffeine and remove most of the interfering components. The SPE method of Lyold et.al [8], Mottaleb et.al [9] and Cho et al.[10]   was used for the extraction of caffeine. The stationary phase was activated with 3mL 50% methanol and pre-equilibrated with 3mL 1% methanol. The columns were air-dried by drawing air through them for 10 minutes. 0.5gm homogenized crude caffeine was dissolved with 100mL double distilled water and then solution (10 mL) was loaded. The adsorbed caffeine was eluted twice with 2mL 046 M Z Abedin, et. al   Determination and Characterization of Caffeine in Tea, Coffee and Soft Drinks by SPE – HPLC    chloroform. The pure caffeine was dried subsequently and then dissolved in another solvent such as toluene and a small amount of petroleum ether was added for recrystallization. In case of HPLC analysis pure caffeine was dissolved in mobile phase. Before HPLC analysis all samples were filtered through a 0.45  m pore size FP 30/45 CA-S filters ( Schleicher and Schuell, Darmstadt, Germany) at 7 bar max. Samples (20  L) of solutions of the samples were injected into the HPLC column.  Method of Spiking: Different amount of black tea samples were spiked with a known amount (0.5g) of analytical standards of caffeine. The standard caffeine solution in water (20mL) was thoroughly mixed with tea solution (after leaching). To ensure homogenization, the spiked samples were shaken by using mechanical shaker for 30min. Then the solvent extraction and SPE processes were carried out as discussed before. The whole process was also carried out for only standard caffeine (0.5g) for the method validation. Unspiked tea samples were also treated similarly. Characterization of pure Caffeine: Different physical methods were employed to characterize the crystalline caffeine.  Determination of melting point: The melting point of different extracted pure samples (after SPE purification) was carried out in a digital melting point apparatus (Electrothermal, IA9100). The average melting points of the samples was 235 0 C. Thin Layer Chromatography: (Whatman, 250   m layer, 20X20 cm) By the purified product (crystalline caffeine) a plate was developed using chloroform as mobile phase and visualized under UV- lamp. The R f   value was measured and it was found 0.63.  IR Spectroscopy: The IR –spectrum of extracted purified caffeine was carried out by using a FT-IR spectroscopy (SHIMADZU, Model-IR Prestige-21). UV Spectrometry:   An UV- absorption spectrum of extracted purified crystalline caffeine was prepared at different absorbance against different wavelength using a UV-absorption spectrophotometer from SHIMADZU Corporation, Japan; Model: UV -1601. The   max  was found to be 275 nm.  HPLC Optimization (calibration):   The different known concentration of caffeine in a solvent, which consisted of 50 mM potassium dihydrogen phosphate (pH=2), acetonitrile and methanol (40:8:2), which was also mobile phase. Standard solutions were injected in HPLC pump by using a syringe after setting proper flow rate (0.5mL/min), attenuation (64) and chart speed (0.5 cm/min) for which a typical pressure is about 1328-1331psi. UV detector was used at a maximum wavelength of 254nm and chromatograms were obtained with almost same retention time. Three replicates of each standard were taken and a plot of relative peak area vs. concentration made to obtain calibration curve.  Determination of caffeine in soft drinks: Each soft drink was degassed properly by placing it in a vacuum flask and connecting the flask to a vacuum pump for 30 minutes. It was kept under vacuum until no more bubbles appear. Then each sample was filtered through a 0.45µm syringe filter with a 5mL syringe. 2 mL of filtered drink sample was 10 times diluted by using mobile phase (solvent). 20 µL of each diluted sample was injected into the column and recorded the trace. The relative peak areas were determined for three replicates of each dilute sample. Then the concentration of each dilute sample and finally the real concentration of caffeine in soft drinks samples were calculated from calibration curve. [11]  Determination of caffeine in Green Tea, Black Tea  and Coffee: 1.0 mg of isolated purified caffeine was dissolved in 100mL mobile phase (solvent). Then the sample was filtered and determined by HPLC as previous section.  Reproducibility: The reproducibility of this method was also checked by determining the percentage recovery of known amount of standard caffeine in the sample. For example if X be the actual content of caffeine in the sample and 5ppm standard caffeine was added then the content become (X+5)ppm and if the observed concentration is X’, then the percentage recovery is given by % Recovery ( ) %1005 ×+′=  X  X    Results & Discussion For preparing calibration curve in HPLC 10 to 60 ppm caffeine standards were used to identify peak at retention time around 6.18-6.22 min. The peak area increases from the lowest standard to the highest. The slope of the curve was 56659, which was used for determination of caffeine in tea, coffee and soft drinks. 047 M Z Abedin, et. al   Determination and Characterization of Caffeine in Tea, Coffee and Soft Drinks by SPE – HPLC            0510152025303540020406080                                                        Figure 1:  Calibration curve for caffeine in 50 mM KH 2 PO 4  (pH=2), acetonitrile and methanol (40:8:2) Table 1: Isolation and Purification of Caffeine from Tea and Coffee. (n=3) Sample Amount of Sample (g) Amount of Caffeine after solvent extraction (g) % of crude Caffeine before SPE Amount of Caffeine after SPE Purification (g) % of Caffeine after SPE Standard Deviation (%) Black Tea 5.0 0.352 7.04 0.167 3.34 0.88 Green Tea 5.0 0.244 4.88 0.122 2.44 0.79 Coffee 1.0 0.137 13.7 0.052 5.20 0.83 Table2:  Percentage recovery of caffeine from black tea spiked with standard caffeine. No. of observations Amount of Black tea (g) Amount of standard Caffeine added (g) Amount of Caffeine obtained after SPE (g) Spiked Result (%) 1 5 0.612 97 2 5 0.610 96 3 4 0.578 96 4 3 0.5 0.545 95 048 M Z Abedin, et. al   Determination and Characterization of Caffeine in Tea, Coffee and Soft Drinks by SPE – HPLC
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