American Journal of Anal yt ical Chemistry, 2011, 2, 411-421
doi:10.4236/ajac.2011.24050 Published Online August 2011 (http://www.SciRP.org/journal/ajac)
An Economical Method for Preparative Purification of
Five Alkaloids from Coptis chinensis Franch by High-Speed
Counter-Current Chromatography Using
Singled Prepared Solvent System by GC
Lianhong Yin, Lina Xu, Xiaona Wang, Binan Lu, Yingnan Li, Mingming Hu,
Yuetao Liu, Jinyong Peng*
College of Pharmacy, Dalian Medical University, Dalian, China
Received August 8, 2010; revised February 22, 2011; accepted May 23, 2011
Abstract
Coptis chinensis Franch, a widely used Traditional Chinese Medicine, shows various kinds of bioactivity.
The major active components of the herb are considered to be alkaloids. Thus, preparative separation of
these alkaloids is critical important for further pharmacology and mechanism studies. In the paper, five alka-
loids from C. chinensis were purified by HSCCC using the solvent system composed of chloroform-metha-
nol-water (2:1:1, v/v/v) single prepared. The content of each solvent in solvent system were determined by
gas chromatography (GC), then according the ratios of solvents in each phase to prepare the mobile and sta-
tionary phase respectively. And a comparative study was carried out between together preparation and single
preparation of the solvent system. The purities and recoveries of all the products were over 98.5% and 92%.
However, 134 mL chloroform, 336 mL methanol and 452 mL water were saved when the two phase were
singled by GC. Our research showed an economical method for separating alkaloids from C. chinensis by
HSCCC using the solvent system single prepared by GC.
Keywords: Alkaloid, Coptis chinensis Franch, High Speed Counter-Current Chromaotgraphy,
Gas Chromatography, Solvent System
1. Introduction
Coptis chinensis Franch (Huanglian in Chinese), a fa-
mous traditional Chinese medicine in China, has been
used for centuries to treat many kinds of disorders, such
as dysentery, cholera, hypertension, leukemia, inflamma-
tion and lung disease [1-3]. The major bioactive compo-
nents of the herb are considered to be alkaloids including
palmatine, berberine, epiberberine, jatrorrhizine and cop-
tisine [4]. Pharmacological studies and clinical practice
have demonstrated that these alkaloids have antibacterial,
anti-inflammatory, anticancer and anti-HIV effects [5-7].
Thus, preparative separation of the alkaloids from the C.
chinensis is critical important for further pharmacology
and mechanism studies. Some methods such as silica gel,
polyamide, preparative RPLC and high-speed counter-
current chromatogramphy (HSCCC) have been reported
previously. But the conventional column chromatogra-
phy is tedious and requiring multiple chromatography
steps, preparative RPLC have a smaller load ranges
[8,9]. In addition, the adsorptions on stationary phase
material and the solvents consume in both of two
methods are serious. Thus, HSCCC as a good effective,
high recovery and big load sample method is expected
and used frequently to separate the alkaloids in recent
years.
HSCCC, a unique continuous liquid-liquid partition
chromatographic technique [10-12], allows directly in-
troduction of crude samples into the column without
pre-preparation, and yields a highly efficient separation
in several hours, when sample load ranges from milli-
grams to kilograms [13,14]. It also can purify some
compounds with an excellent sample recovery, which are
having similar polarities [15,16]. As an efficient liquid
chromatography preparative technique, HSCCC has a
great potential for separation and purification of various

L. H. YIN ET AL. 412
natural products, and it has been widely used in many
fields, such as plant activity constituents, ocean biologic
activity constituents, antibiotic, polypeptides, protein and
chiral components and so on [17-21].
Yang et al. [22] have used an optimum solvent system
to separate four alkaloids including palmatine, berberine,
epiberberine and coptisine from C. chinensis by prepara-
tive HSCCC, successfully. Peng et al. [23] also separated
four alkaloids with high recoveries from C. chinensis by
HSCCC with direct injection the powders of a raw mate-
rials without any preparation. It was not difficult to find
that only four alkaloids were purified, and the purities of
some products were not high enough. And for the long
separation time, large amount of mobile phases were
needed to finish the separations. Thus, a new condition
of HSCCC and an economical method of single prepara-
tion of solvent system need to be developed for the five
alkaloids separation from C. chinensis.
Selection of the solvent system is the most important
factor for successful separation by HSCCC. We can se-
lect suitable solvent systems by looking at previous sys-
tems according to the chemical structures of the com-
pounds using a literature search. We also can determine
the partition coefficients (K, K = Cs/Cm, where Cs is the
concentration of the sample in the stationary phase, and
Cm is the concentration in the mobile phase) of the
chemicals, when the solvent system producing good K
values in the range of 0.5 to 2 can be selected. After se-
lection suitable solvent system in general HSCCC pro-
cedure, the stationary and mobile phases of the solvent
system are required to be prepared. The conventional
method for preparation of the two-phase solvent system
is as follows: according to the volume ratio of the se-
lected solvent system, suitable volumes of the solvents
are all added into a separated funnel and the mixture is
thoroughly equilibrated at room temperature, and the
upper and lower phases are separated shortly before use.
In HSCCC, the volume of mobile phase required is ac-
cording to the flow rate and separating time, and the use
of stationary phase needed is only on basis of the
HSCCC column size. Thus, when above preparation
method is used, there will be much stationary phase is
surplus, which is not only hostile to our environment
protection, but also waste the resources.
The solvent system often used in HSCCC is composed
of several kinds of organic solvents, so the composition
of the upper and lower phases in solvent system can be
analyzed by gas chromatography (GC) [24,25]. Thus,
each layer could be prepared separately as required vol-
ume for minimizing solvent wastage [26]. In the present
paper, the economical method was used for the single
preparation of solvent system. Five alkaloids including
palmatine, berberine, epiberberine, jatrorrhizine and cop-
tisine (Figure 1) from C. chinensis were successfully
purified by HSCCC with the suitable volume of the sta-
tionary and mobile phases single prepared by above eco-
nomical method. And a comparison of the purities and
recoveries of the products, separation time, and the use
of the selected solvents with the HSCCC separation us-
ing the solvent system together prepared by conventional
method was carried out. The advantage of our research
was that an economical method was established for
separating alkaloids from C. chinensis, more compounds
were separated and more solvents were saved.
Figure 1. The chemical structures of the five alkaloids.

L. H. YIN ET AL.413
2. Experimental
2.1. Plant Materials and Chemicals
C. chinensis was purchased from a local drug store (Da-
lian, China) and authenticated by Dr. Yunpeng Diao
(Dalian Medical University, Dalian, China). The stan-
dards including palmatine, berberine, epiberberine, cop-
tisine and jatrorrhizine were all purchased from the Chi-
nese National Institute of Control of Pharmaceutical and
Biological Products, Beijing, China.
Methanol used for HPLC was chromatographic grade
(TEDIA, USA). Phosphoric acid and all other organic
solvents used for sample preparation or HSCCC separa-
tion were analytical grade and purchased from ShenLian
Chemical Factory (Shenyang, China). Reverse osmosis
Milli-Q water (18 MΩ) (Millipore, USA) was used for
all solutions and dilutions.
2.2. Apparatus
The HSCCC instrument used in this study was a
TBE-300A high-speed counter-current chromatograph
(Shanghai Tauto Biotech Co., Ltd., Shanghai, China)
with three multilayer coil separation columns connected
in series (I.D. of the tubing = 1.6 mm, total volume = 260
mL) and a 20 mL sample loop. The revolution radius or
the distance between the holder axis and central axis of
the centrifuge (R) was 5 cm, and the β values of the mul-
tilayer coil varied from 0.5 at the internal terminal to 0.8
at the external terminal (β = r/R, where r is the distance
from the coil to the holder shaft). The revolution speed of
the instrument could be regulated with a speed controller
in the range between 0 and 999 rpm. The HSCCC system
was equipped with a model TBP-50A constant-flow
pump (Shanghai Tauto Biological Company, China), a
UV-Vis detector (Model UV-8823B, Beijing, China),
and a model N2000 workstation (Zhejiang University,
Hangzhou, China). The experimental temperature was
adjusted by HX 1050 constant temperature circulating
implement (Beijing Boyikang Lab Implement, Beijing,
China).
The HPLC analyses were performed on an Agilent
1200 system (Agilent Technologies, Waldbronn, Ger-
many), equipped with 1322A online vacuum degasser,
G1311A quaternary pump, G1329A autosampler, G1314B
UV detector and Chemstation software (Agilent techno-
logies).
The solvent analysis was carried out with GC-14C
(SHIMADZU, Japan) equipped with FID detector and a
capillary column (30 m × 0.32 mm, i.d. 0.25 μm film
thickness).
2.3. Preparation of the Crude Extracts and
Sample Solution
The pulverized plant material of 800 g was extracted
three times with 70% aqueous ethanol (solvent: sample
ratio = 10:1, v/w) under reflux and 4 h for each. The fil-
trate was collected and evaporated under reduced pres-
sure at 60˚C, and the residue was obtained. Then, the
residue was re-dissolved in water, which was adjusted to
pH 2.0 by hydrogen chloride and stayed for one night.
After that, the solution was filtered and the pH value was
adjusted 10.0 with NaOH solution. Subsequently, the
solution was extracted by chloroform, and the under-
layer was separated and evaporated under reduced pres-
sure at 40˚C to dryness. Deep yellow power was pro-
duced, which was lyophilized and stored in a refrigerator
for further HSCCC isolation.
The sample solution was prepared by dissolving 300
mg crude extract in 20 mL of the solvent mixture con-
sisting of equal volumes of stationary and mobile phases
single or together prepared of the solvent system com-
posed of chloroform-methanol-water (2:1:1, v/v/v).
2.4. The Single Preparation of two-Phase Solvent
System used in HSCCC
In this study, we applied GC to single prepare the sta-
tionary and mobile phases of the solvent system used in
HSCCC. The stationary and mobile phases of the se-
lected solvent system were first prepared together by the
traditional method. Then the contents of chloroform and
methanol in the two phases were analyzed by GC using
n-butanol as the internal standard and acetone as the dis-
solvent.
In the paper, different internal standards, capillary
columns and temperature programs were investigated to
optimize GC chromatographic conditions. Finally, a
good separation for peaks of chloroform, methanol and
n-butanol was achieved when the separation carried out
on the following conditions: GC equipped with FID de-
tector and a capillary column (30 m × 0.32 mm, i.d. 0.25
μm film thickness) was used; the column temperature
was held at 30˚C for 10 min, and then programmed to
120˚C at 10˚C/min. The injector and detector tempera-
tures were set at 150˚C and 180˚C, respectively, and in-
jections were made in the split mode with a split ratio 1:
20. Nitrogen was used as the carrier gas. The contents of
chloroform and methanol in the stationary and mobile
phases were determined individually, and suitable vol-
umes of each solvent were calculated for single prepara-
tion of the stationary and mobile phases.
First, the correlations between the volumes and the
weights of the stationary and mobile phases of the se-

L. H. YIN ET AL. 414
lected solvent system were investigated, and we found
that excellent calibration curves were existed. The linear
ranges (mL), regression equations (Y = aX + b) and R2 of
each phase were obtained compared with their weights.
For stationary phase, in the range of 8.7 ~ 400 mL, Y =
0.9262X + 0.1727 (R2 = 0.9999); for mobile phase, in the
range of 11.4 ~ 1500 mL, Y = 1.3612X + 0.9451 (R2 =
0.9999), in which Y is the weight of stationary phase or
mobile phase and X means the volume of each phase.
In GC analysis, the standard solution containing chlo-
roform, methanol and internal standard at the concentra-
tions of 0.02942, 0.01582 and 0.04040 g/mL, respec-
tively, was prepared, which was used for subsequent
determination. Then, 20 mL stationary and mobile phases
were measured with the weights of 18.6967 and 28.1691
g, respectively, and dissolved in acetone to prepare sam-
ple solutions. The internal standard was also added to
afford the concentration of 0.04040 g/mL. All the solu-
tions mentioned above were analyzed by GC, and the
peak areas were recorded. The concentrations (g/mL) of
chloroform and methanol in each phase were calculated
according to the following formulae (1) and (2) [27].
Thus, the concentrations of chloroform and methanol
were 0.03535 g/mL and 0.41203 g/mL in the stationary
phase, and 1.28125 g/mL and 0.09390 g/mL in the mo-
bile phase, respectively. So, according to the formula (3),
the concentrations of water in the two phases were
0.48746 g/ mL and 0.03331 g/mL, respectively.
SS
a
R
R
A
C
f
A
C
 (1)
''
X
Xa
SS
A
Cf
A
C
 (2)
where, a
f
is the correction factor; S
A
is the peak area
of internal standard;
R
A
is the peak area of standard;
S is the concentration of internal standard; C
R
C is the
concentration of standard; X
A
is the peak area of ana-
lyte in test sample; X is the concentration of analyte
in test sample;
C
'
S
A
and are the peak area and con-
centration of internal standard in the test sample.
'
S
C
WTC
CCCC
M
(3)
where, W is the concentration of water in one phase;
Tis the density of one phase (g/ml), C and
C
C C
M
Care
the concentrations of chloroform and methanol in one
phase, respectively.
For the known density of each solvent, we can switch
the concentration of each solvent (g/mL) X to volume
ratio (mL/mL) X. In stationary phase, the volume ra-
tios of chloroform, methanol and water were 0.02403,
0.52090 and 0.48746 mL/mL; and in mobile phase, the
volume ratios were 0.87100, 0.11870 and 0.03331
mL/mL, respectively
C
T
Then, according to the formula (4), we can calculate
the volumes of chloroform, methanol and water in the
stationary and mobile phases.
X
VVTX (4)
where, is the total volume of the stationary and mo-
bile phases needed in HSCCC; X
V is the volume of
solvent needed to prepare the stationary or mobile phase.
V
2.5. The Rraditional together Preparation of
two-phase Solvent System used in HSCCC
The HSCCC experiments were performed with a two-
phase solvent system composed of chloroform-metha-
nol-water (2:1:1, v/v/v). Two methods for preparation of
the stationary and mobile phases were carried out. One,
the stationary and mobile phases were prepared together
by the conventional method. After thoroughly equili-
brating, the two phases of the solvent mixtures in a sepa-
rating funnel at room temperatures were separated
shortly before use. The other, the stationary and mobile
phases were single prepared, according to the results of
GC analysis, namely 1102 mL chloroform, 150 mL
methanol and 42 mL water were added together to pre-
pare the mobile phase, and 6 mL chloroform, 135 mL
methanol and 127 mL water were mixed to prepare the
stationary phase.
2.6. HSCCC Separation Procedure
In each separation, the multiplayer coil column was first
entirely filled with the upper phase as the stationary
phase. Then the mobile phase was pumped into the
‘head’-end of the HSCCC coil column at a suitable flow
rate of 1.5 mL/min, while the HSCCC apparatus was
rotated at a speed of 800 rpm. After a clear mobile phase
was eluted from the tail outlet and the two phases had
established a hydrodynamic equilibrium throughout the
column, the sample solution was injected through the
injection valve. The effluent from the outlet of the col-
umn was continuously monitored at 280 nm and each
peak fraction was collected according to the elution pro-
file. After the separation was completed, the centrifuge
was stopped and retention of the stationary phase was
measured by collecting the column contents by forcing
them out of the column with pressurized gas.
2.7. HPLC Analysis for Purity Determination
In the present paper, the crude sample and each HSCCC
peak fraction were all analyzed by HPLC. The analysis
was carried out on an Agilent Eclipse Plus C18 (150 mm
× 4.6 mm, i.d., 5 μm), and the mobile phase composed of
methanol (A)-3% phosphoric acid (B) (pH 2.0, adjusted

L. H. YIN ET AL.415
by triethylamine) with a gradient mode (0 ~ 15 min,
25%A→30%A; 25 ~ 38 min, 30%A; 38 ~ 60 min,
30%A→35%A). The flow rate was controlled at 0.8
mL/min, and the detection wavelength was selected at
270 nm.
2.8. Chemical Structure Identification and
Confirmation
Identification of the HSCCC peak fractions was carried
out by MS (API 3200 mass spectrometer, Applied Bio-
systems, USA), UV spectra (U-3010 UV, Hitachi, Japan),
and the standards.
3. Results and Discussion
3.1. Optimization Suitable HPLC Analytical
Conditions
A sensitive method for determination of the alkaloids in
crude extract of C. chinensis is a prerequisite to the
HSCCC separation. In recent years, many methods have
been developed to detect the alkaloids in C. chinensis,
such as capillary electrophoresis (CE), LC-MS and
LC-MS-MS [28-34]. Among them, CE analysis is high
sensitive and excellent, but the pre-condition and back-
ground solution are complex, and the technique can not
be controlled easily. LC-MS and LC-MS-MS technique
are fast, sensitive and accurate, but the apparatus is too
expensive and not all research departments can afford it.
By now, there are many HPLC analytical methods have
been reported to determine alkaloids in C. chinensis
[35,36]. However, we can easily find that some flaws
were existed in reported papers including long separation
time, not good separation, complex mobile phase and the
limited number of detected compounds. In the present
paper, different chromatographic column packed with
different materials purchased from different companies,
mobile phases composed of acetonitrile-water and metha-
nol-water with some modifiers including phosphoric
buffer, acetic acid, formic acid, phosphoric acid, formic
acid adjusted by ammonia or by triethylamine with dif-
ferent pH values, and different gradient elution modes
were all investigated. Good separation was achieved
when the process was carried out on an Agilent Eclipse
Plus C18 (150 mm × 4.6 mm, i.d., 5 μm), and the mobile
phase composed of methanol (A)-3% phosphoric acid (B)
(pH 2.0, adjusted by triethylamine) with a gradient elu-
tion mode (0 ~ 15 min, 25%A→30%A; 25 ~ 38 min,
30%A; 38 ~ 60 min, 30%A→35%A). The detection
wavelength was selected at 270 nm and the flow rate was
controlled at 0.8 mL/min. On the optimized conditions,
the beautiful chromatograms of crude sample and mixed
standards are shown in Figure 2(a) and Figure 2(b), in
which the peaks 1, 2, 3, 4 and 5 correspond to coptisine,
epiberberine, jatrorrhizine, berberine and palmatine, re-
spectively. The advantage of the method was that the
mobile phase was simple and no salt or buffer was
needed. The separated time was mediate, and all the al-
kaloids were separated fully.
3.2. Optimization of HSCCC Conditions
According to HPLC analysis shown in Figure 2(a),
there are five major components in the crude extract of C.
chinensis. Many papers have been reported to separate
alkaloids from this plant by HSCCC [22,23]. The solvent
system composed of chloroform–methanol–water is con-
sidered to be one of the classical solvent systems in al-
kaloids purification, but the ratios between the solvents
were not invariable. The crude extracts of Sophora fla-
vescens Ait. were separated and purified by high-speed
counter-current chromatography (HSCCC) with a two-
phase solvent system composed of chloroform-metha-
nol-2.3 × 10−2 M NaH2PO4 (27.5:20:12.5, v/v/v) [37],
and the optimum solvent systems CHCl3-MeOH-0.3
M/0.2 M HCl (4:1.5:2, v/v/v) were used to separate lap-
paconitine, ranaconitine, N-deacetyllappaconitine and
N-deacetylranaconitine from Aconitum sinomontanum
Nakai [38]. So, in our research, the solvent systems com-
posed of chloroform-methanol-water at the volume ratios
of 4:3:2 (v/v/v) and 2:1:1 (v/v/v) and chloroform-etha-
nol-water at the volume ratios of 4:3:2 (v/v/v) and 2:1:1
(v/v/v) were further optimized. The stationary and mo-
bile phases of those four kinds of solvent systems were
all prepared by together and single preparation methods,
and the K-values of five compounds were determined.
The results are listed in Table 1. It was obvious that the
K-values of the compounds epiberberine, coptisine and
jatrorrhizine, and the compounds palmatine and berber-
ine had no difference when the solvent system composed
of chloroform-ethanol-water (2:1:1, v/v/v) was used as
the two-phase solvent system in spite of the methods of
preparation. When chloroform-ethanol-water (4:3:2,
v/v/v) or chloroform-methanol-water (4:3:2, v/v/v) were
used as the separation systems, the targets could not be
well separated and the purities of the compounds were
not satisfactory. Thus, they were not suitable for HSCCC
separation of the five alkaloids from the crude extract of
C. chinensis. The other solvent system tested in present
study was chloroform-methanol-water (2:1:1, v/v/v), and
suitable K-values were produced not only in condition of
together preparation of the stationary and mobile phases,
but also in condition of single preparation method. Thus,
the solvent system composed of chloroform-methanol-
water (2:1:1, v/v/v) was selected to purify the targets

L. H. YIN ET AL. 416
(a)
(b)
Figure 2. The HPLC chromatograms of the crude sample from C. chinensis Franch and the standards. (a) The crude extract;
(b) The mixed standards. Peak 1, 2, 3, 4 and 5 correspond to coptisine, epiberberine, jatrorrhizine, berberine, and palmatine,
respectively.
Table 1. The K-values of the targets in different solvent systems together or single prepared.
The K-values of the targets
Solvent system (v/v) Preparation
method Coptisine EpiberberineJatrorrhizine Berberine Palmatine
1a 2.60 2.16 4.53 1.48 0.97
Chloroform-methanol-water
(2:1:1) 2b 2.44 1.95 4.29 1.83 1.28
1 3.00 2.36 2.52 1.93 1.36 Chloroform-methanol-water
(4:3:2) 2 1.98 1.39 1.89 1.08 0.84
1 1.42 1.30 1.32 0.71 0.67
Chloroform-ethanol-water
(2:1:1) 2 1.24 1.15 1.23 0.65 0.63
1 2.14 1.89 2.02 1.24 1.05
Chloroform-ethanol-water
(4:3:2) 2 2.63 2.45 2.03 1.51 1.30
a Together preparation of the stationary and mobile phases; b Single preparation of the stationary and mobile phases.
from the crude extract of C. chinensis.
Other HSCCC conditions, such as flow rate of mobile
phase, separation temperature and revolution speed, were
also important for a successful HSCCC separation. The
flow rate of the mobile phase determines the separation
time, the amount of stationary phase retained in the col-

L. H. YIN ET AL.417
2
umn, and therefore the peak resolution. The different
temperatures may cause different retentions of the sta-
tionary phases, and the speed may produce the volume of
the stationary phase retained leading to lower peak reso-
lution or excessive sample band broadening by violent
pulsation of the column. So, different flow rates of the
mobile phase, different temperatures and different revo-
lution speeds were investigated. Finally, we found that
the flow rate of mobile phase set at 1.5 mL/min, the
temperature sated at 30˚C, and the apparatus rotated at
800 rpm were the best conditions for this HSCCC
separation.
Under the optimized conditions, the crude extract was
successfully separated by HSCCC using the solvent sys-
tem composed of chloroform-methanol-water (2:1:1,
v/v/v). Figure 3(a) shows the chromatogram of the crude
extract purified by HSCCC when the stationary and mo-
bile phases of the solvent system were single prepared,
and Figure 3(b) shows the chromatogram of the crude
extract isolated by HSCCC when the stationary and mo-
bile phases of the solvent system were together prepared.
Five fractions (, , , and ) were collected aⅠⅡⅢⅣ Ⅴc-
cording to the profiles shown in Figure 3(a) and Figure
3(b), respectively.
3.3. Purity and Recovery Retermination
The obtained fractions from HSCCC were all analyzed
by HPLC compared with the standards for purity deter-
mination. The outcomes of the contents of the five alka-
loids in crude sample, the purity, and recovery of the
obtained targets are listed in Table 2. It was obvious that,
in the separation performed by HSCCC using single
prepared stationary and mobile phases, the purities and
recoveries of the five alkaloids were over 98% and 92%,
respectively. In the process performed by HSCCC using
together prepared stationary and mobile phases, the puri-
ties and recoveries of the products had no differences
compared with single preparation method. Namely, the
purities and recoveries of the obtained compounds were
good, no matter which preparation method of solvent
system were used.
3.4. Chemical Structure Identification
The obtained fractions, , , and were identⅠⅡⅢⅣ Ⅴi-
fied by MS, UV and the standards. The retention times of
the separated compounds were the same compared with
the standards. The MS and UV spectra data are agree-
ment with those in the literature [4]. Thus, the obtained
fractions , , , and were identified to be paⅠⅡⅢⅣⅤl-
matine, berberine, epiberberine, coptisine and jatrorrhiz-
ine, respectively.
3.5. Comparison Study of Single and Together
Preparation of the Stationary and Mobile
Phases
Through the HSCCC chromatograms and HPLC analysis,
we can easily find that there was no difference between
the stationary and mobile phases of the selected solvent
system prepared together and single. In HSCCC separa-
tion using single prepared stationary and mobile phases,
the retention of the stationary phase was 70%, and the
separation time was 800 min and total of 1265 mL of the
mobile phase was required, which was calculated as the
following formula (5), and 260 mL stationary phase was
required only on basis of the HSCCC column size.
1tmp
VVV

 (5)
where Vtmp means the total volume of the mobile phase
required in HSCCC separation; V1 is the volume of the
stationary phase eluted out from the column for hydro-
dynamic equilibrium, and V2 is the use of the mobile
phase to elute the targets, which was calculated accord-
ing to the formula (6), where
M
Tis separation time and
M
F
is the flow rate of the mobile phase.
2
M
M
VT F

 (6)
The compositions of the mobile phase and stationary
phase were analyzed by GC to calculate the solvent con-
sume. According to the volume of each phase used, the
solvent used in the procedure was obtained. Thus, total
of 1108 mL chloroform, 285 mL methanol and 169 mL
water were needed in HSCCC procedure with single
preparation method.
In the separation procedure using together prepared
stationary and mobile phases, the retention of the sta-
tionary phase was 77%, and the separation time was 820
min, and total of 1290 mL mobile phase was needed,
which was also calculated with the formulae (5) and (6).
260 mL stationary phase was also required. The solvent
system composed of chloroform-methanol-water (2:1:1,
v/v/v) was accurately prepared to obtain the required
mobile and stationary phases. There were three calibra-
tion curves established to describe the correlations be-
tween the use of the mobile phase with chloroform,
methanol and water volumes. The linearity of the solvent
volume (X, mL) and the mobile phase volume (Y, mL)
was investigated in the range of 15 - 1500 mL. Y =
1.0372X + 1.6264 (R2= 0.9999, for chloroform); Y =
2.0743X + 1.6264 (R2 = 0.9999, for methanol); Y =
2.0743X + 1.6264 (R2 = 0.9999, for water). Thus, 1242
mL chloroform, 621 mL methanol and 621 mL water
were required to prepare the solvent system according to
the calibration curves, when the stationary and mobile
phases were prepared together.
Thus, 134 mL chloroform, 336 mL methanol and 452

L. H. YIN ET AL. 418
(a)
(b)
Figure 3. The HSCCC chromatograms of the crude extract from C. chinensis Franch. HSCCC conditions: Two-phase solvent
system: Chloroform-methanol-water (2:1:1, v/v/v); flow rate: 1.5 mL/min; revolution speed: 800 rpm; detection wavelength:
280 nm; sample size: 300 mg; separation temperature: 30˚C. Ⅰ, , , and ⅡⅢⅣ Ⅴare the collected fractions. (a) the station-
ary and mobile phases are single prepared by GC; (b) the stationary and mobile phases are together prepared by the conven-
tional method.

L. H. YIN ET AL. 419
Table 2. The detailed information for separating the five alkaloids from C. chinensis Franch by HSCCC using different sol-
vent system preparation methods.
Together preparation method Single preparation method
Compound Contenta Amountb Recoveryc Purity Amount Recovery Purity
Palmatine 7.42% 20.8 mg 92.07% 98.53% 21.3 mg 95.13% 99.42%
Berberine 13.00% 38.1 mg 96.00% 98.27% 37.4 mg 95.51% 99.60%
Epiberberine 5.70% 16.3 mg 94.05% 98.67% 16.4 mg 94.71% 98.75%
Coptisine 8.84% 25.4 mg 95.12% 99.31% 25.1 mg 93.81% 99.12%
Jatrorrhizine 1.31% 3.7 mg 93.98% 99.82% 3.7 mg 93.50% 99.31%
aThe content of the component in the crude extract of C. chinensis Franch; bThe amount of the component separated by HSCCC;
c22
Re cov(%)100%
11
PW
ery PW






. Where P1 is the content of the compound in crude sample; W1 is the amount of the crude sample for
HSCCC isolation; P2 is the purity of the obtained compound; W2 is the amount of the obtained compound from HSCCC. P1 and P2 were cal-
culated by external standard method of HPLC.
Table 3. The details of the HSCCC separation with different preparative methods and the expenditures of the three solvents.
Preparation
methoda
Retention of the
stationary phase
Separation
time
Stationary phase
requiredb
Mobile phase
requiredc
Required of the
two phases Chloroform MethanolWater
Mobile phase ()
1m
V1242 mL 621 mL 621 mL
Together
preparation 77% 820 min 260 mL 1290 mL Stationary phased ()
1
s
V0 mL 0 mL 0 mL
Total volume
()
11tms
VV V
1
1242 mL 621 mL 621 mL
Mobile phase ()
2m
V1102 mL 150 mL 42 mL
Single prepa-
ration 75% 800 min 260 mL 1265 mL
)Stationary phase (2
s
V6 mL 135 mL 127 mL
Total volume
()
22tms
VV V
2
1108 mL 285 mL 169 mL
Saved reagent
()
12St t
VVV 134 mL 336 mL 452 mL
aThe Preparation method means the method to prepare the stationary and mobile phases in HSCCC process; bThe use of the stationary phase in HSCCC is the
column volume of the apparatus; cThe use of the mobile phase in HSCCC process is calculated according to the flow rate of the mobile phase and the separation
time; dThe stationary phase and mobile phase are prepared together.
mL water were saved when the stationary and mobile
phases were single prepared in HSCCC purification. All
the details are shown in Table 3.
4. Conclusions
In this paper, five alkaloids from C. chinensis were suc-
cessfully separated by HSCCC using the solvent system
composed of chloroform-methanol-water (2:1:1, v/v/v),
of which the stationary and mobile phases were singled
prepared by GC. The results indicated that the method
for the alkaloids separation is applied, and the single
preparation technique of the two phases by GC in
HSCCC was efficient and solvent saving. And an eco-
nomical method was established for separating alkaloids
from C. chinensis. The single preparation of solvent sys-
tem by GC might be widely used in long time HSCCC
separation and purification.
5. Acknowledgements
This research was partially supported by the Key Labo-
ratory of Liaoning University (No. 2008S072), Liaoning,
China.
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