SEPARATION OF INDIVIDUAL CATECHINS FROM GREEN

TEA USING SILICA GEL COLUMN CHROMATOGRAPHY

AND HPLC
RYSZARD AMAROWICZ', FEREIDOON SHAHIDI 2 . 3 and
WIESLAW WICZKOWSKI'
'Department of Food Sciences
Institute of Animal Reproduction and Food Research
Polish Academy of Sciences
ul. Tuwima 10
P. 0.Box 55
10- 718 Olsztyn. Poland

'Department of Biochemistry
Memorial University of Newfoundland
St. John S,NL, A l B 3x9, Canada

Received for Publication December 18,2002

Accepted for Publication February 20,2003

ABSTRACT

Crude catechin mixturesfrom green tea were separated into sixfractions using
a silica gel column chromatography and a chloroform-methanol-water (65:35:10,
v/v/v, lower phase) solvent system. Fraction I was free of catechins, fraction II
contained epicatechin (EC), fraction 111 had epicatechin and epigallocatechin
(EGC),fraction IVpossessed EGC,fraction V contained EGC, epicatechin gallate
(ECG) and epigallocatechin gallate (EGCG). andfiaction VI had EGCG. EC and
EGC were separatedfrom fractions II, 111and IVusing HPLC with a RP-18 semi-
preparative column and a water-dimethylformamide-methanol-acetic acid
(I57:40:2: 1, v/v/v/v) solvent system. For isolation of EGC, ECG and EGCGfiom
fractions V and VI a water-acetonitrile-methanol-acetic acid (159:36:4:1, v/v/v/v)
solvent system was employed. Chemical structures of purlfied catechins were
further confirmed by ESI-MS.

INTRODUCTION

Carechins are the main polyphenols of grean tea. The chemical structures of four
principal catechins found in green tea, namely (-) epicatechin (EC), (-)
epigallocatechin(EGC),(-)epicatechin-3-gallate(ECG), and (-)epigallocatechin-3-
gallate (EGCG) are shown in Fig. 1.

'Corresponding author. TEL: 709-737-8552; FAX: 709-737-4000; EMAIL: fshahidi@mun.ca

Journal of Food Lipids lO(2003) 165-177. AIIRIghts Resewed.

0Copyrighi by Food C? Nuirition Press, Inc.. Trumbull, Connecticut. 165
166 R. AMAROWICZ, F. SHAHIDI and W. WICZKOWSKI

Strong biological activity of catechins has been a main reason for extensive
studies of this topic. The catechins possess bacteriostatic (Fukai ef al. 1991) and
antimutagenic (Wang ef al. 1989;Yen and Chen 1994; 1995)properties. They may
inhibit the activity of HIV reversed transcriptase (Nakane and Ono 1990) and
protect against halitosis and dental caries (Ui et al. 1991; Sakanaka et af. 1992).
Consumption of tea on a regular basis has been associated with reduced risk of
several forms of cancer in human populations (Bushman 1998; Dreosti et af. 1997;
Hollman et al. 1999). Antioxidant properties of catechins have been confirmed in
several studies using different model systems (Terao et al. 1994; Amarowicz and
Shahidi 1995a; Chen and Ho 1995; Nanjo et al. 1996; Roeding-Penman and
Gordon 1997; Balentine ef al. 1997; Guo ef al. 1999).
HPLC is a major tool for separation of individual catechins. However,
application of this method requires high content of separated compound in the
solution that is injected into semi-preparative HPLC column. The low pressure
column chromatography before HPLC is also necessary. Sephadex LH-20
chromatography has most commonly been used for this purpose (Hoefler and
Coggon 1976; Amarowicz and Shahidi 1995a, b; Chen and Ho 1995). Application
of sillica gel column chromatographyand semi-preparativeHPLC for separation of
individual catechins from green tea was the aim of the present study.

MATERIALS AND METHODS

Materials

All solvents used were HPLC grade. Methanol, chloroform, dimethyl-

formamide, acetonitrile, ethanol, acetic acid, and hydrochloric acid were acquired
from the Fisher Scientific Company (Nepean, ON) and vanillin from Sigma
Chemical Co. (St. Louis, MO). Chinese green tea leaves were obtained from Anhui
Province of the Peoples Republic of China.

Extraction of Crude Catechins

A crude mixture of catechinswas extracted from 50 g green tea leaves using 500
mL of hot water (80C) over a 1 h period (Price and Spitzer 1993) and partially
purified by employing a counter-current chromatography using water-chloroform
(1 :1, v/v) and water-ethyl acetate (1:1, v/v) solvent systems (Matsuzaki and Hara
1985).
 167
SEPARATION OF GREEN TEA CATECHINS ON SILICA GEL

Column Chromatography

One gram of crude catechins was dissolved in 10 mL of methanol and applied

onto a silica gel (Merck, Dannstad, Germany) chromatographic column (3.0 x 60
cm) equilibrated with the lower phase of a chloroform-methanol-water(65:35: 10,
v/v/v) solvent mixture (Tanizawa et al. 1983). The same solvent was used for
elution. Fraction (88, each 20 mL) were collected using a fraction collector. The
absorbance of eluate from each tube was measured at 280 nm. In addition,
absorbance at 500 nm was read after color development reaction for catechins
(Price et al. 1978). According to the absorbance values eluates were pooled into 6
fractions, solvent was evaporated and dried residues weighted. UV spectra of
individual fractions, dissolved in methanol, were recorded using a Hewlett Packard

FIG. 1. CHEMICAL STRUCTURES OF GREEN TEA CATECHINS

8452A diode array spectrophotorneter.Fractions separated were also examined on

silica gel TLC plates (Sigma, St. Louis, MO) using the same solvent as that for
column chromatography.Vanillin-hydrochloricacidreagent (Karchesy et al. 1989)
was used for color development reaction of catechins.
168 R. AMAROWICZ, F. SHAHIDI and W. WICZKOWSKI

Semi-preparative HPLC

Individual catechins from silica gel column chromatographic fractions were

separated using HF'LC. A Shimadzu chromatographic system (Kyoto, Japan)
consistingof a LCdA pump, SPD-6AVW-VIS spectrophotometricdetector, SCL-
6B system controller, CR 50 1 Chromatopac,semi-preparativeHibar prepacked RT
column (250 x 10 mm) with Lichrosorb RP-18 (7 im) (Merck, Darmstad, Germany)
was used. The mobile phases were: A (for fractions 11, 111, and IV) water-
dimethylformamide-methanol-acetic acid (1 57:40:2: 1, vlvlvlv) (Hoefler and
Coggon 1976 ) and B (for fractions V and VI) water-acetonitrile-methanol-acetic
acid (79.5: 18:2:0.5,v/v/v/v) (Saijo 1982); and flow rates were: 4 mL / min (phase
A) and 3 mL / min (phase B) using an injection volume of 500 pL.

TLC and Analytical HPLC

Pure catechins so obtained were examined by TLC and HPLC using an

analytical column. Conditions of TLC were the same as those for fractions after
silica gel column chromatography. For analytical HPLC the chromatographic
system was as described above. An analytical CSL column (250 mm x 4.4 mm) with
Spherisorb-ODS-2(10 pm ) (Chromatography Sciences Company Inc., Montreal,
Canada) was used. Flow rates were 0.8 mL (phase A) and 1.2 mL / min (phase B)
using an injection volume of 20 pL. For preparative and analytical HPLC, detector
response was monitored at 280 nm.

ESI-MS

Electrospray Ionization mass spectra (in negative mode) of individual catechins

dissolved in 80% methanol (v/v) were obtained using a Shimadzu liquid
chromatography-mass spectrometry unit LC MS - QP 8000 (Kyoto, Japan). The
condition of analysis were as follows. The curve desolvation line (CDL)
temperature: 240C; CDL voltage: - 50 V; probe voltage: - 3.5kV, defragmentation
voltage: - 45 V; nebulizer gas flow of 2.8 mllmin, flow rate of 0.2 mL/min and
injection loop was 10 pL.

RESULTS AND DISCUSSION

Chromatographic separation of individual catechins along with the amount of

each fraction is shown in Fig. 2. Results presented in Fig. 2 and 3 indicate that silica
gel column chromatography does not assure complete separation of individual
catechins. Color reactions of eluates with vanillin as well as TLC results confirmed
the absence of catechins in fraction I. However, this fraction contained some color
 169
SEPARATION OF GREEN TEA CATECHINS ON SILICA GEL

compounds that had a maximum absorbance at 408,505,536,608 and 664 nm (Fig.

3 , 4 , and 5 and Table 1).
The presence of catechins was confirmed in fractions 11-VI. TLC analysis
showed one spot for fractions 11, IV and VI, two distinct spots for fraction I11 and
two joined spots for fraction V (Fig. 5). Fractions 11-VI did not show any absorption
in the visible region, but exhibited maximum UV absorbance at 274,276 at 282 nm,
respectively. Shoulders of these fractions were observed between 330 and 360 nm
(Fig. 4 and Table 1).
TABLE 1.
UV-VIS SPECTRAL DATA OF FRACTIONS OF CRUDE CATECHINS SEPARATED ON A
SILICA GEL COLUMN

I 272,408,506,536,608,664 3 14,378,468
n 282 330,360
111 282 356
Iv 274 346
V 276 352
VI 276 3 60

i n I
EC EC EGC EGC EGC ECG EGCG
I
EGCG

FIG. 2. SCHEME FOR SEPARATION OF INDIVIDUAL GREEN TEA CATECHINS

170 R. AMAROWICZ, F. SHAHIDI and W. WICZKOWSKI

Tube number ( 20 ml / tube )

Fractions : I+ I I ~ I I I t I v Vt + VI 4
FIG. 3. SEPARATION OF CRUDE CATECHMS ON A SILICA GEL COLUMN

Semi-preparativeHPLC chromatogramsusing a water-dimethylformamid-acetic

acid solvent system (Fig. 6) showed that fractions I1 and 111 contained a catechin
with retention time of 10.42 min and fractions I11 and IV contained a catechin with
retention time of 20.5 1 min. Using the solvent system water acetonitrile-methano1-
acetic acid, three catechins with retention times of 7.05, 10.11 and 16.60 min were
separated from fraction V and one catechin with retention time of 10.1 1 min from
fraction V1 (Fig. 7). The high purity of the isolated catechins was confirmed by
analytical HPLC. No additional peaks from impurities were observed (Fig. 8).
The chemical structure of the four purified catechins were confirmed by ESI-
MS in negative mode. The spectra are displayed Fig. 9. The ions with m/s of 289,
305,441 and 457 were assigned to [M-Hl-ofseparated EC, EGC, ECG, and EGCG,
respectively. Thus, from the semi-preparativeHPLC results of fractions from silica
gel it was possible to ascertain that EC was present in fraction I1 and 111, EGC in
fractions 111, IV and V, ECG in fraction V, and EGCG in fractions V and VI.
 171
SEPARATION OF GREEN TEA CATECHNS ON SILICA GEL

0
E
0
0
P

0
0
a

0
0
VI

3
3
d

0
F?

0
0
N

3
%

3
0

< P

0
0
\D

0
2

0
0
d

0
4

3
0
PI
172

1.0 -

0.5 - 00
0°8 0

- - - - - - -
I I1 III IV v VI

FIG. 5. TLC CHROMATOGRAM OF FRACTIONS SEPARATED ON A SILICA GEL COLUMN

EGC
EC

I . . " " " ' * ~ ' " ' " '

10 20 30 10 20 30 10 20
Time ( min ) Time ( min ) Time ( min 1

FIG. 6 , SEMI-PREPARATIVE HPLC CHROMATOGRAM OF FRACTIONS 11,111, and IV

SEPARATED ON A SILICA GEL COLUMN
Mobile phase water-dimethylformamide-methanol-acetic acid (1 57:40:2:1, vlvlvlv)
 173
SEPARATION OF GREEN TEA CATECHINS ON SILICA GEL
174 R. AMAROWICZ, F. SHAHIDI and W. WICZKOWSKI

EGC

ECG

I I 1 I 1 I I

20 40 60
Time (min)

FIG. 8. ANALYTICAL HPLC CHROMATOGRAM OF A MIXTURE OF PURE CATECHINS

OBTAINED FROM SILICA GEL COLUMN FOLLOWED BY SEMI-PREPARATIVE HPLC
SEPARATION AND MIXING
Mobile phase water-dimethylformamide-methanol-acetic acid (1 57:40:2:1, v/v/v/v)

The method used in this study is much faster and more economical than those
which employ Sephadex LH-20 column chromatographyprior to semi-preparative
HPLC. Silica gel column after washing with methanol may be used several times
for chromatographicseparation of catechins. In the present method I .8 L ofmobiie
phase was used for silica gel column chromatography.For SephadexLH-20 column
chromatography of crude catechins 1.8 L of chloroform-methanol-petroleum
(1:2:1, v/v/v) or 560 mL of ethanol were necessary (Amarowicz and Shahidi
1995a, b). Saijo (1982) used approximately 4 L of 40% acetone or 4.6 L of
methanol-hexane-ethyl acetate-water (1000:30:25:5, v/v/v/v) for separation of
catechins on a Sephadex LH-20 column.
A sufficient separation of ECG on a silica gel column allows use of water-
acetonitrile-methano1-aceticacid mobile phase for semi-preparativeHPLC. Using
this system the retention time for ECG was only 16.60 min. Hoefler and Coggon
(1976) reported a retention time of 30 min for ECG using an RP-18 analytical
column with water-dimethylformamide-methanol-aceticacid (157:40:2:1, v/v/v/v).
 175
SEPARATION OF GREEN TEA CATECHINS ON SILICA GEL

15
'00: EC
' O 0 1 EGC
- 80:
a,
V
C
m 60
U
c
3
n
m
w 40
.->
-
Y
m
20

*
245

100 200 3 10 400 500

m/z

'"3 ECG
44 1

'O01 EGCG
7

- 80 -5 80
169
8
5
U
60
c
3
9
m
w 40
.-> 3 9 40
.-c
I
m -
m :

100 200 300 400 500 100 200 300 400

4e
500

FIG. 9. ESI-MS SPECTRA OF SEPARATED CATECHINS

For a preparative column with mobile phase water-acetone-tetrahydrofuran

(78:12:10, v/v/v) retention time for ECG was 50 min (Matsuzaki and Hara 1985).
Thus, in terms of 'solvent volume' time, and the cost of column material silica gel
column separation offers a viable alternative for isolation of pure catechins, in
conjunction with HPLC.
176 R. AMAROWICZ, F. SHAHIDI and W. WICZKOWSKI

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