Food Chemistry 131 (2012) 1539–1545

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Food Chemistry

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Analytical Methods

Column-chromatographic extraction and separation of polyphenols, caffeineand theanine from green tea

Li Wang a,1, Li-Hong Gong a,1, Chang-Jian Chen a, Han-Bing Han a,b, Hai-Hang Li a,⇑a Guangdong Provincial Key Lab of Biotechnology for Plant Development and College of Life Sciences, South China Normal University, Guangzhou 510631, Chinab College of Chemistry and Life Sciences, Guangdong University of Petrochemical Technology, 139 Guandu Road 2, Maoming City, Guangdong 525000, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 10 January 2011Received in revised form 24 September2011Accepted 28 September 2011Available online 5 October 2011

Keywords:Green teaColumn-chromatographic extractionColumn separationPolyphenolsCaffeineTheanine

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.09.129

⇑ Corresponding author. Tel./fax: +86 20 8521 2630E-mail address: li_haihang@yahoo.com (H.-H. Li).

1 Authors contributed equally to this work.

A highly efficient column-chromatographic extraction (CCE) followed by sequential adsorption to extractand separate bioactive compounds from green tea was developed. Tea powder was loaded into columnswith 4-fold solvents and eluted through a cyclic CCE. High-quality tea extracts with greater than 90%extraction efficiencies of polyphenols, epigallocatechin-3-gallate, caffeine, theanine and polysaccharideswere obtained with 4-fold water circulated five times among different columns at 70 �C. Similar results,except for low polysaccharide extraction (35.5%), were obtained with 4-fold 30% ethanol circulated threetimes at room temperature. The highly concentrated water extraction was directly passed through col-umns of polyamide, DM130 macroporous and 732 ion exchange resins, resulting in high-purity polyphe-nols (99%), caffeine (98%) and theanine (98%) after simple purification of the eluates from each column.This method uses simple equipment, minimum solvents and can be used for both quantitative analysisand continuous preparation of high-quality tea extracts and bioactive compounds.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Tea is the most widely consumed beverage in the world afterwater. It contains substantial amounts of polyphenols (specificallyepigallocatechin-3-gallate or EGCG), caffeine, theanine, polysac-charides and other compounds that have unique biological activi-ties and health benefits (Bolling, Chen, & Blumberg, 2009; deMejia, Ramirez-Mares, & Puangpraphant, 2003; Hauber, Hohen-berg, Holstermann, Hunstein, & Hauber, 2009). Approximately3.5 million tons of commercial tea is consumed worldwide eachyear, with about 1.0 million tons, or 30%, being low-quality andwaste tea from tea processing and garden maintenance. Thelow-quality and waste tea contain the same kinds of bioactivecompounds as regular tea and in similar amounts. Additionally,summer and autumn tea varieties, which are not good for drinkingdirectly, contain more polyphenols and caffeine than high-qualityspring tea and are a significant source of natural health-promotingcompounds (Li, Gong, & Zhang, 2007). To develop simple andhighly efficient methods for the extraction and separation of bioac-tive compounds from tea is critical for the economical and compre-hensive utilization of tea resources.

ll rights reserved.

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The extraction and analysis of polyphenols and other bioactivecompounds have been studied extensively (Beecher, Warden, &Merken, 1999; Guillarme, Casetta, Bicchi, & Veuthey, 2010).Although supercritical CO2 extraction is an efficient method to ex-tract bioactive compounds from tea, the high equipment and pro-duction costs limit its application. Industrial tea extraction ismainly based on the maceration method combined with stirring,circulation, ultrasonic, microwave or enzymatic treatment (Kwang& Sang, 2008; Li, Huang, Tang, & Deng, 2010; Perva-Uzunalic et al.,2006; Turkmen, Velioglu, Sari, & Polat, 2007). These methods re-quire large volumes of solvent, have high equipment and energycosts and have relatively low extraction efficiencies.

Several methods have been applied to the separation of poly-phenols and other bioactive compounds from tea, including frac-tionation with organic solvents, precipitation with inorganic ionsand resin adsorption. The first two methods require large amountsof organic solvents or inorganic salts and do not generate goodyields or the separation of polyphenols and caffeine, while resinadsorption has been reported to be an effective method of separat-ing polyphenols. Our group (Ouyang et al., 2009; Tang, Zhou,Zhong, & Zhu, 2003) have reported that polyamide resin showssignificant selective adsorption of polyphenols over caffeine andseparates them efficiently. Lin et al. (2004) reported that 732 cat-ion exchange resin showed good adsorption of theanine at lowpH and high desorption at high pH and concluded that this resinis good for the separation and purification of theanine.

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We have reported a simple and highly efficient column-chro-matographic extraction (CCE) method for the extraction and prep-aration of cordycepin from waste medium of Cordycep militaris (Ni,Zhou, Li, & Huang, 2009). Here, using this new method, we success-fully extracted polyphenols, EGCG, caffeine, theanine and polysac-charides from green tea at high extraction efficiencies with either a4-fold excess of water at 70 �C or 30% ethanol at room temperature.The extraction solution, which has a high concentration of targetcompounds with few impurities, is then directly passed throughthree adsorptive columns of polyamide, macroporous and cationexchange resins. Highly purified polyphenols, caffeine and thea-nine can be obtained after simple purification of the eluates fromeach of the adsorptive columns. The developed method uses simpleequipment, minimum volumes of extraction solvents and few con-centration steps, and this method can be used for the environmen-tally friendly production and quantitative analysis of high-qualitytea extracts and their bioactive compounds.

2. Materials and methods

2.1. Green tea material and reagents

The green tea material used was the low-quality by-product oftea processing and was provided by the Yingde Tea Factory of theGuangdong Provincial Institute of Tea. The tea material was driedat 65 �C for 4 h and then ground and sieved; material between833 lm and 350 lm was used for experiments.

Standard compounds of EGCG, caffeine and theanine were pur-chased from Sigma (St. Louis, MO, USA). HPLC-grade solvents werepurchased from Burdick & Jackson Inc. (Muskegon, MI, USA). Poly-amide, DM130 macroporous and 732 cation exchange resins werepurchased from Shandong Chemical Co. (Shandong, China). Food-grade ethanol (95%) was used in all extractions and purifications.Other analytical- or biochemical-grade organic solvents and chem-ical reagents were purchased from local suppliers.

2.2. Macerating extraction of bioactive compounds from green tea

For macerating extraction as a control for traditional extractionmethods, 5 g of tea powder was extracted with a 10-fold excessvolume of solvents (w/v) by 100-W ultrasonic treatment for 1 h,unless otherwise stated. The extraction solution was filteredthrough double layers of filter papers or centrifuged at 5000g for10 min and used for the chemical determination of componentsor filtered through a 0.45-lm membrane filter for HPLC analysis.

2.3. Column-chromatographic extraction of active compounds fromgreen tea

CCE was performed in glass chromatographic columns as de-scribed previously (Ni et al., 2009). For CCE, solvents of 4-fold vol-ume of the tea powder weight were loaded onto the column(1.5 cm diameter), and then tea powder (8 g unless otherwise sta-ted) was gradually added and allowed to sink freely down into thesolvent. After 1 h, the column was eluted with fresh solvents asabove by common procedures at a flow rate of 2 bed volumes(BVs)/h, and eluates were collected in fractions, each with a solventvolume equal to 4 times the sample weight (w/v). Target com-pounds in each fraction were then analysed.

For cyclic CCE, only the first fraction of eluates was collected asthe final extraction solution. The second fraction was used to ex-tract the next tea material. All fractions thereafter were used forsequentially eluting the next column. Through the cyclic CCEmethod, the total volume of final extracting solution was only 4

times the weight of the dry material (v/w), while the columns wereeluted three or more times.

2.4. Column-chromatographic separation of active compounds

All resins were pretreated sequentially with 1 mol/L HCl, 1 mol/L NaOH and 95% ethanol for 4 h or more and washed extensivelywith water after each pretreatment. After the final pretreatment,resins were loaded into columns and washed with distilled wateruntil the eluates were free of ethanol. Three consecutive columnextractions were performed independently to adsorb and separatepolyphenols, caffeine and theanine. The extraction solution was di-rectly passed over the following columns: the polyamide columnto adsorb polyphenols, the macroporous resin column to adsorbcaffeine and the 732 cation exchange resin column to adsorb thea-nine. Each column was washed with distilled water, and then thecompounds were eluted. The resins were reused for five timeswithout significant decrease in performance after the same pre-treatment method, as described above.

2.5. Determination of compound concentrations

Total polyphenols were extracted and analysed according to theNational Standard Method of the China National Accreditation Ser-vice for Conformity, which was used as a control method to refer-ence the subsequent extraction efficiencies. Three grams of teamaterial was extracted in 450 mL boiling water for 45 min. Afterfiltering, the residues were extracted twice with 100 mL water.The polyphenols, EGCG, caffeine, polysaccharide and theanine con-centrations in the combined extraction solution or tea materialwere determined and used as the baseline standard.

The concentrations of the polyphenols were determined withthe ferrous tartrate colorimetric method (GB/T8313–2002). Theconcentration of EGCG, the major polyphenols, was monitored byHPLC as described below to represent itself as well as total poly-phenols in some processes.

To determine the polysaccharide concentration, tea extractswere concentrated to 1/10 of the initial volume, and 1.5 mL con-centrated extracts was mixed with 8 mL 95% ethanol. After stand-ing at 4 �C for 5 h, the solution was separated by centrifugation at5000g for 20 min, and the pellet was re-dissolved in 1.5 mL dis-tilled water and used to determine the polysaccharide concentra-tion by the phenol sulphate colorimetric method.

A Shimadzu SPD-20A HPLC system (Shimadzu, Japan) with LC-20AT UV detector, YMC-packed ODS column (250 mm � 4.6 mm,5 lm) and analytical software was used for the analysis of EGCG,caffeine and theanine in all samples. EGCG and caffeine were ana-lysed by HPLC, with water:methanol:acetic acid (73:25:2, v/v/v) asthe mobile phase and UV detection at 280 nm; theanine was ana-lysed with a 0.05% trifluoroacetic acid aqueous solution as the mo-bile phase and UV detection at 203 nm. All samples were filteredthrough 0.45-lm membrane filters before injection into the HPLCapparatus. The sample peaks were identified by their retentiontimes and co-injection tests with their corresponding standardcompounds, and the quantitative analysis of each compound wasperformed using the peak area based on the corresponding stan-dard curves.

Ethanol concentrations are expressed in volume percentage (%,v/v). Extraction efficiencies and products purities of all substancesare expressed in mass percentage (%, m/m). All experiments wererepeated at least three times, and the results are given as the meanof three independent experiments ± standard error. Duncan’s testat the 0.05 probability level was used to determine the differencesamong treatments.

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3. Results and discussion

3.1. Solvent selection for the extraction of bioactive compounds fromgreen tea

Green tea contains multiple bioactive compounds, includingpolyphenols (primarily EGCG), caffeine, theanine and polysaccha-rides. It is important to have a one-step method to extract all ofthese compounds for the production of high-quality tea extracts.We have reported a highly efficient CCE procedure for the extrac-tion of compounds from biological materials. The method consistsof two steps; dissolving a target substance into the extraction sol-vent and eluting it from the material in the chromatographic col-umn. The key point for the first step is to find good solvents todissolve the target compounds (Ni et al., 2009).

Using the traditional maceration method, different concentra-tions of food-grade ethanol and extraction conditions were testedto optimize the extraction of EGCG and caffeine from green tea.As shown in Fig. 1A, the extraction efficiencies of EGCG and caf-feine showed a rapid increase with ethanol concentrations from0% to 30%, and then a slow increase from 30% to 60% ethanol;the extraction efficiencies decreased when the ethanol concentra-tion was higher than 60%. The best solvent for both EGCG and caf-feine was determined to be 60% ethanol using the macerationmethod.

As water is a widely used solvent in industrial tea extraction,the efficiencies of extracting multiple bioactive compounds usingwater or 60% ethanol were compared. As shown in Fig. 1B, theextraction efficiency of theanine was the same for both solvents,while 60% ethanol showed a much higher extraction efficiencyfor total polyphenols, EGCG and caffeine; polysaccharide, whichis an important bioactive tea component, was not extracted with

Fig. 1. Extraction of bioactive compounds from green tea by the maceration method wEGCG (white) and caffeine (black) with different concentrations of ethanol. (B) extract(EGCG), caffeine (CAF) and theanine with water (white) or 60% ethanol (black). (C–E) Efextraction of TPP, TPS, CAF, EGCG and theanine.

ethanol. Water showed balanced extraction efficiencies of poly-phenols, EGCG, caffeine and polysaccharides, although polyphe-nols, EGCG and caffeine had low extraction efficiencies.

3.2. Column-chromatographic extraction of multiple compounds fromgreen tea with ethanol

The extraction efficiencies of EGCG and caffeine with 30%, 45%and 60% ethanol were compared using the CCE method. As shownin Fig. 2A and B, the three concentrations of ethanol resulted insimilar efficiencies of extraction for both EGCG and caffeine, asboth compounds could be extracted to 95% and 97%, with 8-foldand 12-fold solvent volumes, respectively. This result indicatesthat maximum extraction can be achieved with the CCE methodusing 30% ethanol, while the maceration method requires 60% eth-anol to attain maximum extraction. A lower ethanol concentrationnot only reduces extraction costs but also increases the extractionefficiency of polysaccharides by about 35%, which is described inSection 3.4. Through the cyclic CCE procedure, a 4-fold excess of30% ethanol achieved the EGCG and caffeine extraction efficienciesby greater than 95%, compared to the extraction efficiencies of91.5% for EGCG and 76.5% for caffeine with 10-fold 30% ethanolin the maceration method (Table 1).

3.3. Column-chromatographic extraction of bioactive compounds withwater from green tea

The extraction efficiencies of polyphenols, EGCG and caffeinethrough maceration were lower than 40% when using water atroom temperature (Fig. 1A and B). To improve the extractionefficiencies with water, the effects of maceration time, solventpH and ultrasonic treatment on the extraction of the five groups

ith a 10-fold excess of solvent (v/w) for 1 h at room temperature. (A) Extraction ofion of tea polysaccharides (TPS), tea polyphenols (TPP), epigallocatechin-3-gallatefects of extraction times (C), pH values (D) and ultrasonic treatments (E) on water

Fig. 2. Column-chromatographic extraction (CCE) of EGCG (A, C and E) and caffeine (B, D and F) from green tea. A and B, Extraction with 30% (h), 45% (Q) and 60% (j)ethanol. C and D, Extraction with room temperature water at different height-to-diameter (H/D) ratio (h, H/D 2.5; Q, H/D 5.0; j, H/D 10.0). E and F, Extraction with water atdifferent temperatures. F1–F5 represent Fraction 1 to Fraction 5, respectively, each fraction with a 4-fold excess volume of the tea material. Each fraction was analysed byHPLC.

Table 1Extraction efficiencies of EGCG and caffeine using different methods and conditions.

Extraction method Extraction efficiency (%)

EGCG Caffeine

Maceration extraction10 Volumes of water, RT* 37.5 ± 2.6 a 35.3 ± 2.1 a10 Volumes of water, 70 �C 69.6 ± 3.9 b 65.7 ± 3.6 b10 Volumes of 30% ethanol, RT* 91.5 ± 4.8 cd 76.5 ± 3.2 c

Cycling column chromatographic extractionCycling 5 times with 4 volumes of water, RT* 88.0 ± 3.5 c 86.0 ± 3.1 dCycling 5 times with 4 volumes of water,

70 �C96.1 ± 3.1 d 93.8 ± 4.3 e

Cycling 3 times with 4 volumes of 30%ethanol, RTa

97.1 ± 2.8 d 95.7 ± 4.5 e

a RT, room temperature; values are means ± SD, n = 3. Values followed by thesame letter in the same column are not significantly different (p < 0.05).

Fig. 3. The extraction of bioactive substances from green tea by the CCE method. Aand B, Extraction efficiencies of EGCG and caffeine with water as the solvent at 70 �Cin the lab experiment (8 g of tea material, white bar) and large-scale experiment(200 g of tea material, black bar). F1–F5 represent Fraction 1 to Fraction 5, and eachfraction had a 4-fold excess volume of the tea material. C and D, Analysis ofextraction solutions (C) and dried extracts (D) from green tea by the cyclic CCEmethod. White bars represent extraction with water at 70 �C, and black barsrepresent extraction with 30% ethanol at room temperature.

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of bioactive compounds from green tea were tested. As shown inFig. 1C, macerating for 60 min before eluting the columns in-creased the extraction efficiencies of polyphenols, caffeine, EGCGand theanine by 5%, 10%, 10% and 20%, respectively, but this proce-dure had no significant effect on the extraction of polysaccharides.There were no significant differences among three pH conditions(pH 3.0, 6.0 and 9.0) for the extraction of the five groups of com-pounds, although pH 3.0 produced slightly higher extraction effi-ciencies of theanine and EGCG (Fig. 1D). Ultrasonic treatment for30 min and 60 min increased the extraction efficiencies to varyingdegrees for different compounds, with a 60-min treatment beingbetter than a 30-min treatment (Fig. 1E). Based on the results ofthe maceration tests, the conditions for dissolving target com-pounds with water were acidic water (pH 3.0) for 1 h with ultra-sonic treatment for the following CCE procedure.

The extraction efficiencies of all five groups of compounds werepositively correlated with the height-to-diameter (H/D) ratio of teamaterial in the columns; an increase in the H/D ratio from 2.5 to 10resulted in a 5–15% increase in the extraction efficiencies for differ-ent compounds (Fig. 2C and D).

As shown in Fig. 2E and F, the extraction efficiency of EGCG in-creased when the temperature was increased from 30 �C to 70 �C.The highest EGCG extraction efficiency was reached at 70 �C andwas slightly decreased at 80 �C and 90 �C, while the extraction effi-ciency of caffeine increased from 30 �C to 90 �C. The optimal

temperature for the water extraction of both EGCG and caffeineusing the CCE procedure was 70 �C. The extraction efficiencies ofEGCG and caffeine at that temperature were >97% and 95%,respectively.

Based on the results above, EGCG and caffeine can be effectivelyextracted by water through the CCE method by macerating thematerial at a low pH (pH 3.0) with ultrasonic treatment for60 min before loading the material onto columns with a H/D ratioof 10:1. The column is then eluted five times at 70 �C, each with a4-fold excess of water. The extraction efficiencies of the five groupsof compounds reached 91% or more at both the lab experimentallevel (8 g) and the large-scale extraction level (200 g) (Fig. 3Aand B). Through a cyclic CCE procedure, which circulates theextraction solution five times among different columns, the

Fig. 4. Separation of bioactive compounds of from a tea extraction solution bysequential column-chromatographic adsorption of polyamide (A), macroporous (B)and 732 cation ion exchange resins (C).

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extraction solvent can be reduced to a 4-fold excess of the teamaterial with the same high extraction efficiencies (Table 1). Whilein the control tests by the maceration extraction, less than 70%extraction efficiencies for both EGCG and caffeine were obtainedwith 10-fold water and at the same temperature of 70 �C (Table 1).

Considering the high costs of the equipment and energy to heatthe whole column extraction system to 70 �C, a modifiedprocedure for the CCE method with water was tested. Tea materi-als were pretreated with a 4-fold excess of water (pH 3.0) andultrasonic treatment for 1 h at 70 �C and then loaded onto columnsfor eluting at room temperature. The extraction efficiencies ofEGCG and caffeine were 88% and 86%, respectively, through a cyclicCCE procedure (Table 1). This approach could be a good alternativewith a reasonably good extraction efficiency and low equipmentand energy costs.

3.4. Analysis of extraction products from green tea

With two cyclic CCE procedures using 30% ethanol at room tem-perature or water at 70 �C, green tea extracts were prepared forlarge-scale extraction (25 times larger; 200 g). The extraction effi-ciencies of the five groups of bioactive compounds, either in theextraction solution or as a vacuum-dried powder, were analysed.The extraction efficiencies of EGCG, caffeine, theanine, polyphenolsand polysaccharides in the extraction solution were 98%, 95%, 97%,96%, and 91%, respectively, with 70 �C water as the solvent, whilethe extraction efficiencies were 97%, 94%, 97%, 98% and 35%,respectively, with room-temperature 30% ethanol as the solvent(Fig. 3C). After the extraction solutions were vacuum-dried, theextraction efficiencies of the five group compounds in dried pow-der showed similar results, as shown in the extraction solutions,except for slight decreases in the extraction of polyphenols andtheanine (Fig. 3D).

Analysis of the tea extracts showed a significant difference ofpolysaccharide content between the two extracts produced bythe different extraction solvents and conditions. Only 35% of poly-saccharides were extracted by 30% ethanol, while 91% of polysac-charides were extracted by water. The contents of other bioactivecompounds in the green tea extract powder produced using thetwo solvents showed no significant differences except for lowerpolysaccharides in 30% ethanol extracts (0.53%) than in water ex-tracts (1.39%).

When comparing the two extraction procedures with water and30% ethanol, water extraction required higher temperature andlonger solvent circulation time. However, water is the most popu-lar and economically feasible solvent, and water extraction pro-duced high-quality tea extracts with respect to traditional teadrinks and water-soluble components such as polysaccharides.Furthermore, in most cases the ethanol in the ethanol-basedextraction solutions needs to be removed before the solutions areloaded onto adsorption columns for separation, while water-basedextraction solutions can be directly loaded onto separation col-umns. For the quantitative analysis and preparation of polyphe-nols, caffeine and theanine without the consideration ofpolysaccharides, 30% ethanol is a good solvent in the CCE or cyclicCCE method.

3.5. Separation of polyphenols, caffeine and theanine by columnchromatography

The green tea extraction solution produced by the cyclic CCEmethod using a 4-fold excess of water at 70 �C had high concentra-tions of bioactive compounds and could be directly loaded ontoadsorptive columns for separation without concentration. Threesequential adsorptive columns of polyamide, macroporous and732 cation ion exchange resins were used to absorb and separate

polyphenols, caffeine and theanine, respectively. The polysaccha-ride content is lower in green tea than in other tea materials, suchas old tea leaves and roots, and was not collected in the separationprocess.

After repeated tests, the optimal volumes, loading conditions,washing and eluting steps for each column were determined(Fig. 4). Polyamide resin has a high adsorptive capacity of26.71 mg/mL for polyphenols but a weak capacity for caffeineand theanine (Ouyang et al., 2009). The aqueous green tea extract,with 17.81 mg/mL EGCG, was directly loaded onto a polyamidecolumn at a flow rate of 2 BV/h with a total volume of 15 BV,and EGCG started to leak at the 17th BV. The column was washedwith 11 BVs of water without EGCG leakage. The aqueous elutionfrom the 2nd BV during loading to the 6th BV of the water washingcontained more than 95% of caffeine and theanine, and the solutionwas then passed over the second column (macroporous resin).After washing with water, the polyamide column was eluted with3 BVs of 80% ethanol, which resulted in more than 90% of the EGCGbeing eluted (Fig. 4, upper). The eluates were vacuum-concen-trated at 50 �C to the aqueous solution and fractionated once withan equal volume and then twice with a half-volume of ethyl ace-tate, after which 95.6% of the polyphenols was fractionated tothe organic fraction. The combined ethyl acetate was vacuum-dried, and the powder contained 99% of the total polyphenolsand 78.2% of the EGCG. The total recovery rate of EGCG for thewhole extraction, separation and purification process was 78.4%.

The aqueous eluates containing caffeine and theanine from thepolyamide column were directly loaded onto a DM130 macropo-rous resin column at a flow rate of 2 BVs/h. The macroporous resinhas a caffeine adsorption capacity of 13.0 mg/mL, and 11 BVs of

Fig. 5. Diagram of water extraction of bioactive compounds from green tea by the cyclic CCE method, followed by a three-step adsorptive separation of individualcompounds.

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aqueous eluates from the polyamide column with 2.27 mg/mL caf-feine were loaded onto the macroporous column. The column wasthen washed with 4 BVs of water. The eluates during the loadingand the first 2 BVs of washing with water contained more than95% theanine and were collected for the third column for theadsorption and separation of theanine. Caffeine absorbed in themacroporous column was eluted with 3 BVs of 40% ethanol witha desorption rate of more than 98% (Fig. 4, middle). Caffeine eluateswere vacuum-concentrated to the aqueous fraction and fraction-ated once with an equal volume and twice with half the volumeof chloroform, and 94.7% of the caffeine was fractionated to thechloroform fraction. The combined organic fractions were vac-uum-dried, re-dissolved in hot water and crystallized three timesat 4 �C. The crystallized product was pure white and containedmore than 98% caffeine. The total recovery rate of caffeine was87.7%.

The aqueous eluates containing theanine from the macroporouscolumn were adjusted to pH 4.0 with 1 mol/L HCl and loaded ontothe 732 cation exchange column at a flow rate of 2 BVs/h. The resinhas a theanine adsorption capacity of 10.5 mg/mL and was loadedwith 45 BVs of the solution containing 0.44 mg/mL theanine beforetheanine started to leak at the 48th BV. The column was washedwith 2 BVs of water after loading, and the theanine was eluted with6 BVs of phosphate buffer (pH 9.18). Almost all of the theanine(99%) was in the last 5 BVs of the eluates (Fig. 4, lower), and thetheanine eluates were vacuum-dried and re-dissolved in methanolat 60 �C. After filtration at room temperature to remove insolublecompounds, the methanol solution was crystallized at 4 �C over-night. Pure white crystallized theanine, with purity greater than98%, was obtained after three or four cycles of re-crystallizationin methanol. The overall recovery rate of theanine was 78.8%.

The overall process of the extraction and separation of bioactivecompounds from green tea is shown in Fig. 5, and a continuousproduction line for tea extracts and individual bioactive com-pounds could be designed using this procedure.

4. Conclusion

A highly efficient CCE method and a sequential column-chro-matographic separation method were developed for the extraction

of tea extracts and the separation of bioactive compounds, respec-tively. More than 90% of polyphenols, EGCG, caffeine, theanine andpolysaccharides could be extracted by the cyclic CCE method at70 �C using a 4-fold excess of water circulated five times amongdifferent columns, compared to the less than 70% extraction effi-ciencies through the maceration method with 10-fold solvents atthe same temperature (70 �C, Table 1). Similarly high extractionsof all of the groups of compounds except for polysaccharides(35.5%) could be obtained by the cyclic CCE method using a 4-foldexcess of 30% ethanol circulated three times at room temperature.The water extraction procedure could be used for both the quanti-tative analysis and the large-scale preparation of tea extracts andits bioactive compounds. This process could produce a well-bal-anced tea extract with more water-soluble components such aspolysaccharides, and the water-based extracting solution couldbe directly loaded onto separation columns without further con-centration. The extraction procedure with 30% ethanol could beused for the preparation and quantitative analysis of polyphenols,caffeine and theanine when polysaccharides are not needed.

A three-step sequential adsorptive method was first developedand optimized to separate bioactive compounds from green tea ex-tracts. Aqueous extracts with high concentrations of the com-pounds to be separated could be directly passed through threeadsorption columns of polyamide, macroporous and 732 cationion exchange resins, and polyphenols, caffeine and theanine couldbe separately eluted from their respective columns. After concen-tration, fractionation with organic solvents and crystallization,polyphenols, caffeine and theanine could be obtained with greaterthan 98% purity, and the resins were reusable after regeneration.The overall method of extraction and separation of green tea usessimple equipment, a minimum volume of solvent and minimumsolvent concentration steps, and the method can be used for thelow-cost, large-scale, continuous preparation and the quantitativeanalysis of high-quality tea extracts and individual bioactive com-pounds with high efficiency.

Acknowledgements

This work was supported by the Science and Technology Sup-porting Programs of the Guangzhou Municipal Government (pro-gram number 2008Z1-E591), the Panyu District Science and

L. Wang et al. / Food Chemistry 131 (2012) 1539–1545 1545

Technology Programs of Guangzhou City (program number 2009-T-17-1), the Guangdong Provincial Science and Technology Pro-grams (2011B020310008) and Guangdong Natural Science Fund(10151063101000002, S2011010003368).

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