Journal of the Korean Chemical Society 2007, Vol. 51, No. 2 Printed in the Republic of Korea f p ƒ š w er elk yw ½ Á y*» l, w w ywœw (2006. 12. 5 ) Separation of Caffeine and Catechin Compounds from Green Tea by Quercetin Molecular Imprinted Solid-Phase Extraction Yinzhe Jin and Kyung Ho Row* Center for Advanced Bioseparation Technology, Department of Chemical Engineering, Inha University, 253 Yonghyun-Dong, Nam-Ku, Incheon 402-751, Korea (Received December 5, 2006). š f p ƒ š w er +C, EGC, EGCG elk yw w. Quercetin x, MAA, EGDMA ƒ wš AIBN w MIP w w. er elk yw w» w š,, ƒƒ, k, k : p =90:10(vol.%) w. š j m v C 18 f (5 µm, 250Ü4.6 mm, RS-tech z ), k / (40/60, vol.%) wš 0.5 ml/min w. ƒ š m w, er elk yw ƒw. w f p ƒ š œ w yw ƒ +Cyw w w k ƒ. : ƒ š, š, f p, er, ƒlk yw ABSTRACT. In this work, caffeine and some catechin compounds such as +C, EGC and EGCG were extracted from green tea using quercetin molecular imprinted polymers in solid-phase extraction. For synthesis of MIP, quercetin as the templates, MAA as the monomer, EGDMA as the crosslinker and AIBN as the initiator were used. For extraction of caffeine and catechin compounds from green tea, the solid-phase extractions of a load followed by wash and elution procedures were done with water, methanol and methanol:acetic acid=90:10 (vol.%) as the solvents, respectively. HPLC analysis (C 18 column, 5 µm, 250 4.6 mm) with the mobile phase of methanol:water=40:60 (vol.%) at a flow rate of 0.5 ml/min was adopted for the quantitative determination. By solid-phase extraction, the resolutions of caffeine and some catechin compounds from green tea were increased. The quercetin-mip had higher selectivity to +C compounds. Keywords: Molecular Imprinted Polymer, Solid-phase Extraction, Quercetin, Caffeine, Catechin Compounds ƒ» x w p ƒ š w w» w ƒ w š p ü ey x(template)» k» w». 1-3 ƒ š k k w w ƒ š. p ƒ š ƒ š 4-6 p, 165
166 ½ Á y,, w j š w. ƒ š polymer w wš ƒ w. š ù, yw š,»,». ƒ w š Ÿ w w x ww š w w. ƒ š 7-9 100z w z 8 w z w. ƒ š x 10 k ƒ š f w f z û., j w, w ƒ kw x w ƒ š w j. ƒ š w w» w ƒ ƒ w ww š. x ƒ ƒw x ƒ z ƒ w. f z û ƒ š w š j» ƒ ³ w š ³ewš p w y w -k ƒ». 11 ww x yw k ey w k v ƒ. š e w š j m v w ƒ wwš. š w p w k w û p û k w ƒ š. š ƒ š 12 w w k, w, û w ƒ š. j p ƒ š 13-16 w š w. 17-22 x f p w f p ƒ š w w š w l er elk yw z w, ƒ š k š w. elk yw ƒ w f p x w ƒ š w š w l elkyw e w w. Quercetin, caffeine, (-)epigallocatechin(egc), (+) Catechin (+C), (-)epicatechin (EC), (-)epigallocatechin gallate(egcg), MAA(methacrylic acid) Sigma(ST Louis, MO, U.S.A.) AIBN(2,2'-Azobis(isobutyronitrile)) Junsei Chemical Co. Ltd(Japan) EGDMA(ethylene glycol dimethacrylate) Fluka(Buchs, Switzerland), p (analytical grade) ywœ (Incheon, Korea) w. w. Acetonitrile, chloroform, methanol, acetone HPLC grade Pure Chemical Co. Ltd(Ansan, Korea) w. w rv(division of Millipore, Waters, U.S.A.) vl(ha-0.45 m, Division of Millipore, Waters, U.S.A.) w w z w. x 250 ml v j x f p 1 mmol(338.9 mg) š,» 5 mmol (0.43 g) MAA, ƒ 30 mmol(5.94 g) EGDMA, 0.12 g AIBN, š ww porogen wì. porogen mù p 9 ml k 5 ml wì w. yw q» 10 ³ w yw k x ƒ w w 10 ww. œrv w v j ü œ k š œ k w š k 60 o C w š 24 w w w. w yw œ k w w z g. yw w z 35 µm m w yw j» 35 µm w œ wš e j 4 w w 25-35 µm. k z š ep 200 mg w. ƒ š ep w, methanol: acetic acid=90:10(vol.%) w 3 ml 4z x w. w, k x Journal of the Korean Chemical Society
2007, Vol. 51, No. 2 f p ƒ š w er elk yw 167 w 3 ml 4z w. š, ƒ ƒ, k methanol:acetic acid=90:10(vol.%). 1 ml š ƒ š m k z j m v w p š w. 5g 50 o C 150 ml 4 375 rpm w w. 0.2 µm v l w z 1:10 w š w. x»» HPLC l Waters 600s Multisolvent Delivery System Waters 616 liquid chromatography (Waters Associates, Milford, MA, U.S.A.), Rheodyne injector(20 µl sample loop)ƒ. l l HP Vectra 500 PC e Millennium 3.2 w. UV» 2487 UV dual channel detector(waters, Milford, MA, U.S.A.) w. Methanol:water = 40:60(vol.%) w 0.5 ml/min w UV wavelength 270 nm š w. m er elk yw z š j m v w d w.» 5 g w š w ƒ 0.2 mg/ml š wš 5µl 20 µl v y g vj mw w. er ƒ elk yw Table 1 ùkü. x y ƒƒ j m v j (mauüsec) t v er elk yw v(µl). Table 1. Calibration curve equations of caffeine and some catechin compounds Compounds Equations r 2 EGC y=5 10-6 -1.09 0.98 +C y=7 10-7 -1.19 0.99 Caffeine y=1 10-6 -2.10 0.98 EGCG y=1 10-6 +2.40 0.98 x: peak area (mau*sec) y: volume of compounds in the water (µl) Fig. 1. Chemical structures of quercetin, caffeine and some catechin compounds. (r 2 ) w. n 2 ( ) y i yx ( i ) r 2 i = 1 = 1 --------------------------------, y i ( y i y i ) 2 n i = 1 y i = -------- N xz w, er, +C ((+) Catechin), EGC((-) epigallocatechin), EGCG((-) epigallocatechin gallate) r 2 0.97. x x x y w 3 x d w w 10%. Fig. 1 f p, er elk yw w ùkü. f p yw C 15H 10O 7 š 302.3. f p +C EGC w yw ƒ š w w ƒ w ƒ š k w. Fig. 2 š ep e txw. er elk yw ƒ š k w ù l. š ep m j er EGCGƒ ù š EGC +C p œ ù œ. k š ep š, p œ. k / p w p œ +Cƒ er
168 ½ Á y Fig. 2. Scheme of the solid-phase extraction of caffeine and some catechin compounds processing in quercetin MISPE. Fig. 4. HPLC analysis of water-extract by quercetin MIP. Fig. 3. Chromatogram of the water-extract of caffeine and some catechin compounds from green tea by analytical HPLC. wì. f p ƒ š wì sww txw. Fig. 3 j m v w e r elk yw w j m ù kü. Fig. 3, š ep e š f w, er y w š, ƒ. j m v w elk y w w p ƒ ƒw w,, k w w. w : k = 60:40(vol.%). Fig. 4-6 ƒƒ (Fig. 4), k (Fig. 5), k / p (Fig. 6) š ep m k z j m v w j Fig. 5. HPLC analysis of methanol-extract of caffeine and some catechin compounds by quercetin MIP. Fig. 6. HPLC analysis of methanol/aa-extract by quercetin MIP. m. š ep w w elk yw ùkû. ep m Journal of the Korean Chemical Society
f p ƒ š w er elk yw 169 Table 2. Solid-phase extraction of caffeine and some catechin compounds from green tea on the quercetin MIP Compounds EGC +C Caffeine EGCG Water (µg/g) 0.00 0.00 390.01 399.92 Methanol (µg/g) 543.63 215.72 210.91 111.28 Methanol/AA (µg/g) 0.00 169.17 281.94 0.00 Total (µg/g) 543.63 384.89 882.86 511.20 Unit : amounts of sample/g of green tea k w er EGCGƒ k m k z EGC, +C, er EGCG w. elk yw w ƒ f p š. f p ƒ š w w š ü f p ƒ œ x w w œ elk yw z w g. k p ƒ w œ f p w w ƒ w elk y w w. š e z j m w er w, f p w ƒ +C w. ƒ š k r, elk yw +C w k ƒ. Table 2 š e z j m v w w ùkü t. Fig. 4 ùkü, š ep m w w er EGCG ƒƒ 390.01 399.92 µg/g. k š ep m k w +C 215.72 µg/g 46.55 µg/g. +C 44% ƒ f p ƒ š ƒ ƒ w +C w w. ƒ š, ƒ š w p œ w w ƒ w ¼. er k k / p w ƒƒ 210.91 281.94 µg/g 882.86 µg/g elk yw. EGC š k w 543.63 µg/g. w, EGCG k w ƒƒ 399.92 111.28 µg/g k / p. x, f p yw x w ƒ š, œ w elk yw k j l, +C>EGCG>EGC. EGCG ƒ š š ù. k w EGCG š k w EGC f p ƒ š w ƒ ù y œ š. +C k w k / p w f p ƒ š ƒ x w œ w w œ w x w. +C y EGC x w ƒ š w w elk yw w w š w v ƒ. w w ƒ š w p g ƒ k. f p yw x w ƒ š w w. š ep w l er elk yw w. ƒ elk yw š e z. ƒ š ƒ w w k ƒ elk yw +C w k ƒ š. EGCG ep m w z k š EGC k +C k k / p 2007, Vol. 51, No. 2
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