J

ISSN 1615-9306 · JSSCCJ 43 (8) 1393–1614 (2020) · Vol. 43 · No. 8 · April 2020 · D 10609

JOURNAL OF

S SEPARATION
S SCIENCE 8 20

Methods www.jss-journal.com
Chromatography · Electroseparation

Applications
Biomedicine · Foods · Environment

JSSC_43_8_cover.indd 1 30/04/20 7:23 PM

Received: 18 November 2019 Revised: 8 January 2020 Accepted: 3 February 2020

DOI: 10.1002/jssc.201901174

RESEARCH ARTICLE

Optimization extraction and purification of biological activity

curcumin from Curcuma longa L by high-performance
counter-current chromatography
Yan Pan1 Ronghui Ju1 Xueli Cao2 Hairun Pei2 Tianhao Zheng1
Wei Wang3

1 Beijing Vocational College of Agriculture, Beijing, P. R. China

2 Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University, Beijing, P. R. China
3 Beijing Center for Physical and Chemical Analysis, Beijing, P. R. China

Correspondence
Xueli Cao, Beijing Advanced Innovation
Center for Food Nutrition and Human Health,
Beijing Technology & Business University,
Beijing 100048, P. R. China.
Email: caoxl@th.btbu.edu.cn
Hairun Pei, Beijing Advanced Innovation
Center for Food Nutrition and Human Health,
Beijing Technology & Business University,
Beijing 100048, P. R. China.
Email: peihairun@th.btbu.edu.cn
Funding information
Natural Science Foundation of Beijing,
Grant/Award Number: 2184100; National
Natural Science Fund of China, Grant/Award
Number: 21773014
The extraction condition of curcumin from Curcuma longa L was optimized through
four factors and three levels orthogonal experiment based on the results of single fac-
tor tests. Under the optimal conditions: the concentration of ethanol 80%, extraction
temperature 70◦ C, the ratio of liquid to material 20, and extraction time 3 h, a crude
extract with the yield of curcumin 56.8 mg/g could be obtained. The isolation and
purification of curcuminoids from the crude extract was performed on high perfor-
mance counter current chromatography employing an optimized solvent system n-
hexane/ethyl acetate/methanol/water (2/3/3/1, v/v/v/v). From 97 mg crude sample (in
which the purity of curmumin was 68.56%), 67 mg curmumin, 18 mg demethoxy-
curcumin, and 9.7 mg bisdemethoxycurcumin with a high-performance liquid chro-
matography purity of 98.26, 97.39, and 98.67%, respectively, were obtained within
70 min. The antioxidant activities and cytotoxicity of purified curcumin was com-
parable to that of the commercial product, indicating that the biological activity of
curcumin could be maintained by this method.

KEYWORDS
curcuminoids, curcumin, demethoxycurcumin, high performance counter-current chromatography,
turmeric

1 I N T RO D U C T I O N explored in antiprotozoal [1], antioxidant [2], anticancer

Turmeric, Curcuma longa L., is a well-known herb of the
Zingiberacea family and a natural colorant. As the main
component of Curcuma longa L, curcuminoids have been
Article Related Abbreviations: BDMCC, bisdemethoxycurcumin; CC,
curcumin; DAD, diode-array detector; DMCC, demethoxycurcumin; DPPH,
1, 1-diphenyl-2-picrylhydrazyl; HPCCC, high performance counter-current
chromatography; K, partition coefficient; L-I-H, lower phase as mobile
phase elute in head-to-tail mode.
1586 © 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[3–5], preventing diabetic nephropathy [6], and preventing
Alzheimer’s disease [7]. Curcumin (CC), demethoxycur-
cumin (DMCC), and bisdemethoxycurcumin (BDMCC) are
the three main curcuminoids (Figure 1A), and CC is the
most important active component [8]. Due to the suppression
effect of CC on a crucial event in tumor metastasis-epithelial
mesenchymal transition [4], it has been evaluated in clinical
trials for the treatment of various cancers and liver diseases,
rheumatoid arthritis, infectious diseases, and Alzheimer’s
disease [7]. Although DMCC and BDMCC have similar
www.jss-journal.com J Sep Sci 2020;43:1586–1592.
PAN ET AL. 1587

F I G U R E 1 Structural formulas of the main curcuminoids (A) and HPLC analyses of curcumin, demethoxycurcumin, and
bisdemethoxycurcumin standards (B) and the crude sample from Curcuma longa L (C). Conditions: column: Agilent ZORBAX SB-C18 (4.6 ×
250 mm, 5 μm) was used; column temperature, 35ºC; mobile phase, acetonitrile: H2 O (0.5% acetic acid) = 44: 56 (v/v); flow rate, 1.0 mL/min;
injection volume, 10 μL; detection wavelength: 425 nm. (A) Structural formulas of the main curcuminoids. (B) Three standard samples in which
black dotted line is bisdemethoxycurcumin, red dashed line is demethoxycurcumin and blue solid line is curcumin. (C) HPLC chromatogram of crude
ethanol extract from Curcuma longa L. Peak identities: 1, bisdemethoxycurcumin (10.32%); 2, demethoxycurcumin (19.01%); 3, curcumin (68.56%)

structure with curcumin, the slight differences in structure
make them great different in ant-tumor, anti-oxidation, and
other aspects [9]. However, curcumin extract on the market
is usually a mixture of CC, DMCC, and BDMCC. Therefore,
it is necessary to further isolate and purify curcumin com-
pounds, which is of great significance for the identification
of curcuma, the quality control of curcuma preparations and
the modification of the three curcumins results.
Counter-current chromatography (CCC) [10,11] is a
widely applied method for purification of natural compo-
nents. Each component could be separated according its dis-
tribution coefficients during LLE and centrifugal distribution
prcoess [12]. Compare to conventional column chromatogra-
phy, CCC eliminates irreversible adsorptive loss of samples
onto solid support matrix and avoids devitalization of active
compounds [13,14], which can achieve identical purities and
yields producing matching chromatograms [15,16]. Notably,
CCC has been already successfully used in the purification of
phytosterol [17], fatty acids [18], anthocyans [14], polyphe-
nols [19], flavones [20], and many other natural and synthetic
products [21]. It has been reported that CC could be sepa-
rated by counter-current chromatography [22] or centrifugal
partition chromatography [5], but the solvent system contains
chloroform that is forbidden in China. Recently, with the
development of counter-current chromatography technology,
a new high-performance counter-current chromatography
(HPCCC) equipment with higher separation efficiency has
been developed [16]. HPCCC is a recently developed coun-
tercurrent chromatography that accelerates the separations
complete in minutes rather than hours that generally experi-
enced with HSCCC. Solvent systems that were difficult to be
applied before, can also achieve the separation with improved
retention of the stationary phase, and solvent systems used
in counter-current chromatography are gradually becoming
less toxic. In addition, it needs to point that CC is chemical
instability and is sensitive to pH, temperature, and light
[23]. In this way, the bioactivity of the extracted CC is an
important indicator to evaluate the purification method.
Herein, we developed a more friendly solvent system
composed of n-hexane/ethyl acetate/methanol/water to
directly separate and purify curcumin from crude extract.
Complete separation of curcumin, demethoxycurcumin, and
bisdemethoxycurcumin with high purity could be achieved
efficiently in about 1 h through the optimized solvent sys-
tem. Moreover, the bioactivity (antioxidant activities and
tumor cell cytotoxicity) of purified CC shows no significant
difference with commercial product.
2 M AT E R I A L S A N D M E T H O D S
2.1 Reagents and materials
Curcuma longa L was bought from Tongrentang Chi-
nese Medicine. The curcumin, demethoxycurcumin, and
bisdemethoxycurcumin standard products were obtained
from Sigma–Aldrich (China). Ethyl acetate, n-hexane, and
methanol were purchased from Macklin (AR, Shanghai Mack-
lin Biochemical, China). Ethanol was bought from National
Pharmaceutical (AR, China National Pharmaceutical Group
Corporation, China) and distilled water was used. Chro-
matographic grade methanol used for HPLC analysis was
1588 PAN ET AL.

TABLE 1 Factors and levels for orthogonal test of extraction TABLE 2 Result analysis of L9 (3)4 test
Levels Yields
Variable 1 2 3 Number X1 X2 X3 X4 (mg/g)
X1 Extraction time (h) 1 2 3 1 1 1 1 1 23.6
X2 Extraction temperature (◦ C) 60 70 80 2 1 2 2 2 54.2
X3 The concentration of ethanol (%) 70 80 90 3 1 3 3 3 36.9
X4 The ratio of liquid to material (mL/g) 15 20 25 4 2 1 2 3 45.7
5 2 2 3 1 36.4
6 2 3 1 2 36.7
purchased from Fisher (HPLC, Fisher scientific, USA) and
7 3 1 3 2 38.4
HPLC water was supplied by Milli-Q Plus system (Millipore,
Bedford, USA). HeLa (human cervical cancer cells) cells 8 3 2 1 3 32.7

were obtained from the Cell Resource Center (Peking Union 9 3 3 2 1 48.8
Medical College Headquarters on National Infrastructure of k1 38.233 35.9 31 36.267
Cell Line Resource, NSTT). Cells were cultured in DMEM k2 39.600 41.100 49.567 43.100
(Gibco) supplemented with 10% FBS (Gibco) and 1% peni- k3 39.967 40.800 37.233 38.433
cillin/streptomycin (Gibco) at 37◦ C and 5% CO2 . R 1.732 5.200 18.567 6.833

2.2 Apparatus
TABLE 3 Partition coefficients (K) and separation factors (α) of
The CCC apparatus used in this study was Spectrum HPCCC the three components in four solvent systems
(Dynamic Extraction, UK). Spectrum HPCCC contains two
Solvent system 𝛂
columns, an analytical (0.8 mm i.d. tubing, 22.5 mL) and n-hexane/ethyl
a semi-preparative (1.6 mm i.d., 133.5 mL), and the rota- acetate/methanol/ CC DMCC BDMCC
tion speed was adjustable from 0 to 1600 rpm. The column water (v/v/v/v) (K1 ) (K2 ) (K3 ) K2 /K1 K3 /K2
temperature was controlled by an SH150-1500 constant tem- 1/1/1/1 1.77 1.95 2.11 1.10 1.08
perature regulator (Lab Tec, Beijing, China). A KNAUER 1/3/3/1 0.93 1.16 1.25 1.24 1.07
Smartline HPLC system (Berlin, Germany) containing two 2/3/3/1 0.63 1.05 1.67 1.67 1.59
p-1000 pumps, a UV-2500 detector and a EuroChrom work-
3/3/3/1 0.47 0.59 0.75 1.25 1.27
station was equipped with Spectrum HPCCC. An Agilent
1260 HPLC system (Agilent, USA) with diode-array detec-
tor DAD-10A UV (Agilent, USA) was used in this study. ficient (K) and separation factors (α) of each target com-
The NMR data were obtained on an Aglient DD2 600 MHz ponent. The K value of each component in crude Curcuma
NMR spectrometer (Agilent, USA) with tetramethylsilane as longa L extract was measured by HPLC. Generally, 10 mg of
an internal standard. SpectraMax 190 (Molecular Devices, crude sample was dissolved in the selected two-phase solvents
USA) ELISA was used in this study. (Table 3) with 1 mL of upper phase and 1 mL of lower phase.
After violent shaking and equilibrating the two phases com-
2.3 The orthogonal test pletely, 0.5 mL solution of each phase was taken and analyzed
The extraction of Curcuma longa L. was carried out through a by HPLC. The K values of each target component were calcu-
four-factor, three-level orthogonal test based on the single-test lated from the peak area obtained of the upper phase divided
results. The optimization of the extraction conditions is listed by that of the lower phase. Separation factors (α) represented
in Table 1 with the extraction time (X1 ), extraction temper- the ratio of the K values of two adjacent target components.
ature (X2 ), the concentration of ethanol (X3 ), and the ratio of The multilayer column was filled thoroughly with the upper
liquid to material (X4 ). Table 2 represents the experiment vari- phase (stationary phase), and the mobile phase was then
ables and nine experiment points. The content of curcumin pumped into the Spectrum-CCC column at the flow rate
was determined by external standard method (y = 117675x – of 5 mL/min while the apparatus was rotated at speed of
654802, R2 = 0.9978). 1600 rpm. After the equilibration, the crude extract (97 mg)
dissolved in 2 mL upper phase was loaded into the 133.5 mL
column. Chromatograms were recorded at 425 nm and the
2.4 Counter-current chromatography
peak fractions were collected.
separation and identification
The purity of the three components was analyzed by HPLC,
The selection of two-phase solvent system was the key point and their chemical structures were identified by comparing
for CCC where the separation is based on the partition coef- the retention times with that of standard samples in HPLC and
PAN ET AL.
1 H-NMR analysis. The HPLC conditions were as follows:
column: Agilent ZORBAX SB-C18 (4.6 × 250 mm,
5 μm) used at the temperature of 35◦ C; mobile phase:
acetonitrile:H2 O (0.5% acetic acid) = 44:56 (v/v) at the
flow rate of 1 mL/min; injection volume: 10 μL; detection
wavelength: 425 nm.
2.5 Antioxidant activity
The antioxidant activity of the purified curcumin was deter-
mined using the 1, 1-diphenyl-2-picrylhydrazyl (DPPH)
radical scavenging activity assay [22]. Generally, the puri-
fied curcumin solutions at concentrations of 25, 50, and
100 μg/mL were prepared in tubes, 4 mL DPPH (0.1 mM,
dissolved in methanol) were added into each tube and mixed
thoroughly. The tubes were then incubated without light
at room temperature for 20 min. A control sample was
prepared without any curcuminoids and used for baseline
corrections. The absorbance of sample solutions at 517 nm
was recorded. The radical scavenging activity was expressed
as the inhibition percentage according to formula:
Radical scavenging activity (RSA%)
[( ) ]
= 𝐴control − 𝐴analyte ∕𝐴control × 100% (1)
where A are the absorbance values measured for the test and
control sample, respectively. All analyses were repeated three
times, and the results were expressed as mean ± SD.
2.6 Cell viability
HeLa (human cervical cancer cells) cells were obtained
from the Cell Resource Center (Peking Union Medical
College Headquarters of National Infrastructure of Cell
Line Resource, NSTT). Cells were cultured in DMEM
(Gibco) supplemented with 10% FBS (Gibco) and 1% peni-
cillin/streptomycin (Gibco) at 37◦ C and 5% CO2 [24].
To evaluate the cytotoxicity of extracted curcumin and
standard sample, CCK-8 cell viability was carried out. Basi-
cally, 5–8 × 103 cells were seeded in 96-well plates and incu-
bated overnight for further adherence. Cells were treated with
purified curcumin and standard curcumin with concentrations
ranging from 0 to 20 μg/mL for 24 h. Next, CCK-8 was added
to the culture medium at a tenth of the volume and incubated
at 37◦ C for 1 h. Cells without curcumin treatment were used
as control. The absorbance at 450 nm was measured and cell
viability was calculated as formula:
Cell viability (%) = [(𝐴control − 𝐴analyte )∕𝐴control ] × 100%
(2)
where A is the absorbance of cells treated with or without
CCK-8 at 450 nm. Each concentration of curcumin was
1589
repeated five times, and the results were expressed as
mean ± SD.
3 RESULTS AND DISCUSSION
3.1 HPLC analysis of the standards
and crude samples
Figure 1 represents the HPLC chromatograms of standards
(B) and the crude sample (C) under optimized condition. By
comparing the retention times with that of standard samples,
peaks 1, 2, and 3 in crude sample were identified respec-
tively as bisdemethoxycurcumin, demethoxycurcumin, and
curcumin tentatively, which accounted for 10.32, 19.01, and
68.56% of the crude extract of Curcuma longa L sample.
3.2 Optimization for extraction of curcumin
In order to optimize the extraction conditions, an orthogonal
L9 (3)4 test was designed involved four factors with three vari-
ation levels: X1 (extraction time, 1, 2, 3 h), X2 (extraction tem-
perature, 60, 70, 80◦ C), X3 (the concentration of ethanol, 70,
80, and 90%) and X4 (the ratio of liquid to material, 15, 20,
and 25 mL/g), which are listed in Table 1. From the results
(Table 2), the maximum extraction yield of curcumin was
54.2 mg/g. However, it could not be considered as the best
extraction conditions. Because the coefficients of the three
products are R3 > R4 > R2 > R1 , the influence of extraction
parameters decreased in the order of X3 > X4 > X2 > X1 .
Therefore, it seems that the concentration of ethanol was
found to be the most important parameter in the curcumin
extraction process. In this case, the conditions used to achieve
maximum yield of curcumin were optimized as the concentra-
tion of ethanol 80%, temperature 70◦ C, time 3 h, and liquid
to material ratio 20 mL/g, respectively. Through confirmatory
test, the highest yield of curcumin was 56.8 ± 1.2 mg/g.
3.3 Selection of solvent system and
counter-current chromatography separation
Several prerequisites should be considered. According to the
golden rules in selecting optimum separation conditions, par-
tition coefficients of different solvent systems in the range of
0.5–2 for the target component are important for successful
separation by CCC method [11]. Moreover, the separation fac-
tor between two constituents (α = K1 /K2 , K1 > K2 ) is at least
1.5. In addition, solubility and stability of the analytes in sol-
vent should be taken into consideration at the same time, as
is the same case to the rapid and clear separation of solvent
system into phases. It has been reported that the curcumin
had been separated by n-hexane/chloroform/methanol/water
(5/10/7.5/2.5, v/v/v/v) [22] solvent system. However, due to
the toxicity concern, chloroform was not easily available as
1590 PAN ET AL.

F I G U R E 2 HPCCC chromatograms of the crude sample.

Conditions: column, Spectrum: 133.5 mL; flow rate, 5 mL/min; rotary
speed, 1600 rpm; elution time: 70 min in L-I-H mode; detection
wavelength, 425 nm. F1, F2, and F3 were identified to be curcumin,
demethoxycurcumin, and bisdemethoxycurcumin, respectively

a controlled product in China. In this article, we try to use

ethyl acetate to replace chloroform to achieve the separa-
tion. According to another previous research, the curcumin
could not be separated by solvent system, n-hexane/ethyl
acetate/methanol/water (1/1/1/1, v/v/v/v) [25]. However, our
optimization based on the determination of the K and α val-
ues of the target components, as shown in Table 3, revealed
that n-hexane/ethyl acetate/methanol/water (2/3/3/1, v/v/v/v)
could be a potential solvent system to produce a satisfactory
separation.
Therefore, it was employed to separate curcumin,
demethoxycurcumin, and bisdemethoxycurcumin in the
crude sample on an HPCCC instrument. As a result, the F I G U R E 3 HPLC-DAD analysis of F1, F2, and F3. Conditions:
equipment was more robust efficient than previous HSCCC column, Agilent ZORBAX SB-C18 (4.6 × 250 mm, 5 μm); column
instruments. The CCC elution was operated in L-I-H [14] temperature, 35ºC; mobile phase, acetonitrile:H2 O (0.5% acetic
(lower phase as mobile phase, eluted from inner head) mode acid) = 44:56 (v/v); flow rate, 1.0 mL/min; injection volume, 10 μL;
for 70 min after 97 mg of the crude extract was loaded into detection wavelength, 425 nm. (A) F1, (B) F2, and (C) F3
133.5 mL column. The whole separation was completed
in 70 min rather than 5 hours that is generally experienced
1H NMR (DMSO, 600 MHz) of F1: δ 7.516(d,
with HSCCC [22]. Three peak fractions (67 mg F1, 18 mg
F2, and 9.7 mg F3) could be baseline separated (Figure 2). J = 15.81 Hz, 2H), 7.293(d, J = 1.79 Hz, 2H), 7.123(dd,
Compared with the retention time of standard samples, J = 8.21, 1.81 Hz, 2H), 6.795(d, J = 8.14 Hz, 2H), 6.725(d,
HPLC analysis proved the F1, F2, and F3 were curcumin, J = 15.82 Hz, 2H), 6.031(s, 1H), 3.810(s, 6H). All these data
demethoxycurcumin, and bisdemethoxycurcumin, with the are in agreement with that of curcumin.
1 H NMR (DMSO, 600 MHz) of F2: δ 7.535(t, J = 6.71,
purity 98.26, 97.39, and 98.67%, respectively (Figure 3).
6.71 Hz, 3H), 7.503(d, J = 5.64 Hz, 1H), 7.296(d, J = 1.62 Hz,
1H), 7.116(dd, J = 8.20, 1.65 Hz, 1H), 6.794(dd, J = 8.32,
3.4 Identification of purified target 1.47 Hz, 3H), 6.731(d, J = 15.81 Hz, 1H), 6.662(d,
compounds
J = 15.86 Hz, 1H),6.019(s, 1H), 3.811(s, 3H). All these data
F1, F2, and F3 were further confirmed for their structures by are in agreement with that of demethoxycurcumin.
1 HNMR (Supporting Information Fig. S1). The identity of F1, 1 H NMR (DMSO, 600 MHz) of F3: δ 7.544(s, 2H),

F2, and F3 were assigned as curcumin, demethoxycurcumin, 7.517(d, J = 15.80 Hz, 4H), 6.793(d, J = 8.56 Hz, 4H),
and bisdemethoxycurcumin based on agreement of NMR data 6.665(d, J = 15.83 Hz, 2H), 6.013(s, 1H). All these data are
with literatures [22,26]. in agreement with that of bisdemethoxycurcumin.
PAN ET AL.
F I G U R E 4 The antioxidant activities and the cytotoxicity of curcumin on HeLa cells. (A) DPPH radical scavenging activity (RSA) of curcumin
standard and purified curcumin. (B) CCK-8 analysis of curcumin standard and purified curcumin on HeLa cells after 24 h treatment
3.5 Antioxidant activities and cytotoxicity
As a natural bioactive compound, curcumin is chemically
instable to pH, temperature, and light. Therefore, avoiding
compound inactivation during the purification process is
one of the important indicators to evaluate the purification
method. Here, we compared the antioxidant activities and
tumor cell cytotoxicity of the isolated high purity curcumin
with commercial curcumin product (purity > 95%) to explore
the HPCCC method in maintaining the bioactivity of cur-
cumin. As shown in Figure 4A, DPPH assay displayed similar
capability of extracted curcumin and commercial curcumin
in scavenging DPPH radicals, which is consistent with other
antioxidant profiles of curcuminoids [22,27]. Moreover, phar-
macology studies had confirmed the anticancer effect of cur-
cumin through its effect on protein pathways [28]. As shown
in Figure 4B, as an antitumor drug, Curcumin exhibited cyto-
toxicity with an IC50 value of 4.22 μg/mL in HeLa cells.
Cell viability results suggested no significant difference of
HPCCC purified curcumin and commercial product, which
further confirmed the bioactivity of curcumin was remained
during the HPCCC purification.
4 CONC LU SI ON
Overall, here we have developed a more efficient and
rapid HPCCC method for the separation and purification
of curcumin, demethoxycurcumin, and bisdemethoxycur-
cumin from Curcuma longa L. By using n-hexane/ethyl
acetate/methanol/water (2/3/3/1, v/v/v/v) as a two-phase sol-
vent system, three compounds can be completely separated
with high purity in about 60 min. The purities of the isolated
curcumin, demethoxycurcumin, and bisdemethoxycurcumin
can reach 98.26, 97.39, and 98.67%, respectively, while their
contents in the crude extract were 68.56, 19.01, and 10.32%,
respectively. The antioxidant activity and cytotoxicity of
purified curcumin is comparable to the commercial product,
indicating the method maintain the biological activity of
1591
curcumin. Our results demonstrate that HPCCC elution
method is a feasible method to separate and purify curcumin,
demethoxycurcumin, and bisdemethoxycurcumin from
Curcuma longa L, which is more effective than many other
conventional techniques and has more potential to be applied
for commercial industry.
ACKNOW LEDGEMENTS
This work was supported by grants from Natural Science
Foundation of Beijing (No. 2184100) and the National Nat-
ural Science Fund of China (No. 21773014). We would like
to thank Yueheng Qi and Hang Gao from the Key Labora-
tory of Radio Pharmaceuticals of the Ministry of Education
for help with NMR experiments.
CO N F L I C T O F I N T E R E ST
The authors have declared no conflict of interest.
O RC I D
Xueli Cao https://orcid.org/0000-0001-9160-1511
Hairun Pei https://orcid.org/0000-0002-1682-7964
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S U P P O RT I NG IN FO R M AT I O N
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
How to cite this article: Pan Y, Ju R, Cao X,
Pei H, Zheng T, Wang W. Optimization extrac-
tion and purification of biological activity curcumin
from Curcuma longa L by high-performance counter-
current chromatography. J Sep Sci. 2020;43:1586–
1592. https://doi.org/10.1002/jssc.201901174