Original Acta Chromatographica 27(2015)2, 387–398

Research Paper DOI: 10.1556/AChrom.27.2015.2.13

Recycling Preparative High Performance Liquid

Chromatography for the Separation of
Curcumin from Curcuminoids in
Curcuma Longa L.
S. MOLLAYI1, S. TAMHIDI2, H. HASHEMPOUR3, AND A. GHASSEMPOUR1,*
1Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C. Evin,
Tehran, Iran
2Department of Chemistry, Amirkabir University of Technology, Tehran, Iran
3Phytochemistry Group, Department of Chemistry, Faculty of Science, Azarbaijan Shahid Madani

University, Tabriz, Iran

* E-mail: aghassempour@scientist.com, a-ghassempour@sbu.ac.ir

Summary. Curcumin is the main bioactive constituent of turmeric rhizome. A wide

range of pharmacological activities of curcumin has been reported. There has been grow-
ing interest in the development of preparative purification of curcumin. The objective of
this study is the isolation of curcumin from other components by using recycling prepa-
rative HPLC method from curcuma longa L. The extraction was achieved by using ultra-
sonic system with three different solvents, including acetone, dichloromethane, and
ethanol. After optimization of extraction process, preparative HPLC system was first ap-
plied for separation of curcumin. Because of poor resolution of preparative HPLC, recy-
cled preparative HPLC was applied for separation of curcumin with higher purity up to
five cycles. Recycled preparative HPLC resulted in improvement in separation factors
which led to the increase of curcumin purity up to 99.5%. This method is simple, highly
efficient, environmentally friendly, and has been demonstrated to be effective for prepa-
ration and purification of curcumin from curcuma longa L.

Key Words: curcuma longa L., curcumin, preparative HPLC, recycling preparative HPLC

Introduction
The rhizomes of turmeric (curcuma longa L.) belong to the Zingiberaceae
family, and it is a rich source of polyphenolic compounds called curcumi-
noids. The main components of curcuminoids include curcumin (C), de-
methoxycurcumin (DMC), and bis-demethoxycurcumin (BDMC) (Fig. 1).
These compounds have many physiological properties, but lately, it was re-
ported that curcumin is the main bioactive constituent of turmeric rhizome
and has a wide range of pharmacological activities, including anti-
inflammatory [1, 2], anticancer [1, 3, 4], anti-oxidant [1, 5], anti-angiogenic
[6], and immune modulatory [7]. Another study has also shown that cur-

0231–2522 © 2014 Akadémiai Kiadó, Budapest

388 S. Mollayi et al.

cumin is a potential chemotherapeutic agent for cancer as well as a promis-

ing agent in the treatment and prevention of Alzheimer’s disease [8]. In ad-
dition, it may be effective in treating malaria, prevention of cervical cancer,
and may interfere with the replication of the HIV virus [9-11].

Fig. 1. The chemical structures of curcumin, demethoxycurcumin, and

bisdemetoxycurcumin

The choice of solvents for extraction of curcuminoids is restricted to a

few solvents of defined purity. Considering the solubility of the active con-
stituents, the curcuminoids are poorly soluble in the hydrocarbon solvents.
Organic polar solvents are often used to isolate curcuminoids from Cur-
cuma species. In some studies, 95% ethanol and absolute acetone were the
most efficient in extracting curcumin from the turmeric rhizomes [10]. Also,
results revealed that the curcuminoid content was affected by the type of
solvent. Among some extraction solvents (acetone, chloroform, ethyl ace-
tate, dichloromethane, ethanol with varying polarity), acetone was the most
efficient in extracting curcumin from turmeric rhizomes [12, 13]. Also, the
isolation of curcumin, as well as other curcuminoids from curcuma longa L.,
reported numerous methods including microwave-assisted extraction
[14–16], ultra-sonic extraction [14, 15, 17], soxhlet extraction [18], and super-
critical carbon dioxide-assisted extraction [14]. Microwave-assisted extrac-
tion has a bright prospective in the curcumin extraction with the character-
istics of considerable time saving and high extraction efficiency.
In many cases, preparative high-performance liquid chromatography
(preparative-HPLC) is a method needed to satisfy the purity specifications
Recycling Preparative High Performance Liquid Chromatography 389

required on a routine basis, and it is also an important industrially applied

separation process for the isolation and purification of natural compounds,
pharmaceuticals, and other valuable products [19, 20]. However, for achiev-
ing higher purity and recovery yield, high resolution and selectivity are re-
quired. The high resolution in preparative HPLC is highly dependent on
column length. The elongation of column length is a simple way to get a
good resolution. However, this will lead to an increase in pressure in the
systems and amount of stationary phase in columns. In addition, changing
the length of columns in an industrial place is not available, because of pro-
ducing them in specific dimensions. So, the increase of the columns length
is not economically desirable. The necessity of applying an operating
method that can overcome these limitations while obtaining higher resolu-
tion is attractive. Recycling HPLC is one way of realizing this. A schematic
diagram of a recycling HPLC system is given in Fig. 2. In this technique, the
target compounds return to the column after being in the detector several
times to achieve a higher purification. The benefits of these techniques in-
clude increase of column resolution, higher product purity, and recovery
yield. Also, saving on operating costs may be achieved by necessity to no
fresh solvents in the recycling periods [21, 22].

Fig. 2. A schematic diagram of a recycling HPLC system A) before recycling B) after

recycling

Curcuminoids were isolated by using preparative silica plates from

ground turmeric by Anderson et al. [23]. Other studies reported that the
390 S. Mollayi et al.

preparative separations of individual curcuminoids from turmeric powder

were attempted via pH-zone refining countercurrent chromatography [24]
and high-speed countercurrent chromatography [25]. However, none of the
reports described the isolation of curcumin from curcuminoids thru recy-
cling preparative HPLC.
In our previous study, peptides were isolated from Viola ignobilis by us-
ing preparative and two-dimensional HPLC [26]. Also, in another study,
two-dimensional hydrophilic interaction/reversed-phase liquid chromatog-
raphy was used for the preparative separation of polar and non-polar tax-
anes [27]. In this work, after optimization of the solvent for curcuminoids
extraction and scale up from analytical to preparative HPLC method, we
evaluated the recycling preparative HPLC to isolation of higher pure cur-
cumin from other curcuminoids present in turmeric powder.

Experimental
Materials

Curcuma longa L. was procured from a local market in Tehran, Iran. All
HPLC solvents were purchased from Merck by HPLC grade. Other solvents
which were used for extraction were purchased from Merck by high purity.
The reference standard of curcumin was purchased from Sigma.

Extraction of Curcuminoids by Ultrasonic Method

Turmeric (Curcuma longa L.) powder used for the extraction of curcumin
was procured from a local market in Tehran, Iran. In this extraction method,
based on recent reports [8, 12–17], three kinds of the solvents (acetone, di-
chloromethane, and ethanol) were selected. In this way, approximately 20 g
of sample, taken into a thimble and placed in an ultrasonic apparatus, was
set up with various solvents (acetone, dichloromethane, and ethanol).
About 60 mL of solvent was added and sonicated at room temperatures for
1 hour. Brown extract was then cooled, filtered, concentrated using rotary
evaporator, and finally vacuum suction was applied to get crude dried ex-
tract which was black orange in color. Then, the extract was re-dissolved in
diethyl ether and was stirred for 60 min. The diethyl ether extract was dis-
carded, and the powder was dissolved in methanol for HPLC analysis.
Recycling Preparative High Performance Liquid Chromatography 391

HPLC Analysis of the Crude Extracts

For the HPLC analysis, the Knauer (Germany) with binary HPLC pumps,
diode array detector, column oven and a C18 column (Eruspher, 250 × 4.6
mm, 5 μm, 100 Å) was used. The gradient condition for the HPLC analyzed
started with 40% solvent A (methanol) and raised to 60% for 10 min, main-
tained for 10 min in 60%, and then changed to the initial in the next 2 min,
at a flow rate of 1 mL/min, solvent B was water. The column oven tempera-
ture was set at room temperature. The isocratic method done by 50% sol-
vent A. The wavelength used for detection of HPLC analysis was set at
425 nm.

Preparative HPLC Separation

Reversed-phase preparative and recycling preparative HPLC were carried

out on a Knauer liquid chromatography system equipped with a UV detec-
tor. The chromatographic separation was performed on a C18 column (Eu-
rospher, 250×16 mm, 15 μm, 100 Å). UV detection was carried out at
425 nm. Preparative HPLC separation was performed by injecting 1 mL of
extracts at flow rates of 8, 10, and 12 mL/min. The isocratic method with
50% solvent A (methanol) was used for 100 min. Solvent B was water. In the
preparative HPLC, curcumin was collected between 12.5 and 14 min and in
recycling preparative HPLC between 46 and 48 min. The solvent was dried
by freeze drying to obtain a purified compound.

Results and Discussion

The purpose of this study was to develop the recycling preparative HPLC
method for achieving higher pure curcumin. So, this work was done in four
steps: 1. optimization of type of solvent for extraction of curcuminoids from
curcuma longa L., 2. achieving higher resolution for curcuminoids by ana-
lytical HPLC, 3. scale up of preparative HPLC method for achieving higher
amount of pure curcumin, and 4. development of recycling preparative
HPLC method for improvement resolution and achieving pure curcumin.

Optimization of Type of Solvent for Extraction

Different solvents, including dichloromethane, ethanol, and acetone, were

selected for extraction of curcuminoids from turmeric rhizome. After extrac-
tion of curcuminoids according to section, the extracts were analyzed by
analytical HPLC to estimate the percentage composition of individual cur-
392 S. Mollayi et al.

cuminoids present in the extracts. The identity of each peak was confirmed
by spiking with standards and determination of retention times. The results
are shown in Table I.

Table I. The percentage composition of the individual and total curcuminoids obtained
from different solvent extracts

C (%) DMC (%) BDMC (%) Total (%)

Dichloromethane 75.06 18.10 5.6 98.76

Ethanol 63.00 19.93 15.58 98.51
Acetone 66.81 18.81 12.24 97.86

According to the results in Table I, the percentage of curcumin in di-

chloromethane extract is higher than two other extracts. It seems that most
curcumin in dichloromethane extract may be achieved rather than the oth-
ers. However, when the amount of each curcuminoids was calculated by ex-
ternal standard method, the results showed that acetone extract had more
curcumin than the others (Table II). So, acetone extract can be a good source
for the isolation of individual curcuminoids. On top of that, by using ace-
tone as an extract solvent, a higher amount of curcumin was achieved in
comparison with other solvents. So, acetone extract was selected for separa-
tion of curcumin by preparative HPLC.

Table II. The amount (g/kg) of each curcuminoids in curcuma longa L.

C DMC BDMC Total

Dichloromethane 22.73 5.48 1.69 29.9

Ethanol 25.03 7.91 6.18 39.12
Acetone 29.62 8.34 5.42 43.38

Optimization of Analytical HPLC for Isolation of Curcuminoids

Before doing a large-scale preparative HPLC run with its attendant materi-
als’ expense and labor cost, the separation first should be optimized and
tested on a small scale, if at all possible. If scaling up is to be straightfor-
ward, then the small-scale system should have as many parameters as pos-
sible in common with the large-scale system that will be used. Some of the
potential concerns and points to consider are, ideally, that there should be
Recycling Preparative High Performance Liquid Chromatography 393

Fig. 3. A) Gradient technique for separation of curcuminoids with using methanol and
water as mobile phase solvent. B) Isocratic technique for separation of curcuminoids by
using methanol and water as mobile phases with ratio of 50:50 (v/v). C) Chromatogram
of preparative HPLC separation of curcuminoids by using methanol, water as mobile
phases with ratio of 1:1 (v/v), respectively, sample loading concentration of 1 mg/mL
and flow rate of 10 mL/min

no difference between the analytical LC and preparative LC packing except,

perhaps, for particle size and it is easiest to scale up from one column to an-
other if they differ only in diameter, not in length. In our study, several iso-
cratic and gradient techniques were tested. Firstly, we tried to optimize a
few gradient techniques using methanol, acetonitrile, and water as mobile
phase solvents [28]. As shown in Fig. 3A and Table III, we could not achieve
a good enough resolution by using gradient techniques. So, we had to
394 S. Mollayi et al.

change from gradient technique to isocratic technique. In the isocratic tech-

nique [28], the kind of column (C18, C8, and CN) and solvent, ratio of the
solvents in the mobile phase, concentration of the triethylammonium ace-
tate buffer salts, the pH of the mobile phase, flow rate of mobile phase,
temperature of column, and concentration of sample were optimized.
Therefore, the results showed that the best resolution was achieved when
the amount of water was increased higher than 50% (Fig. 3B). However, in
this condition, the retention time was increased (Table III). So, a 50:50 ratio
was selected for water and methanol in isocratic method. The optimum
condition for good resolution was obtained with C18 column, methanol–
water (50:50 v/v), at pH = 7, flow rate 1 mL/min and without any salts at
room temperature.

Table III. Separation factor and Resolution of BDMC, DMC and C

Method A1 Method B2
α R α R

BDMC 1.08 2.01 1.03 2.04

1.083 2.014 1.033 2.044
DMC
1.07c 2.035 1.076 3.965
C 1.07 2.03 1.07 3.96

1Gradient technique by using methanol and water as mobile phase solvents

2Isocraticmethod by using methanol and water as mobile phases with ratio of
50:50, respectively
3Separation factor between BDMC and DMC
4 Resolution between BDMC and DMC
5 Resolution between C and DMC
6 Separation factor between C and DMC

Scale up of Preparative HPLC Method

According to the results achieved from analytical HPLC, we used isocratic

technique for preparative HPLC. Accordingly, a preparative HPLC column
was chosen with the same bed length but about 12 times higher than the
volume of the analytical column; the ratio of cross-sectional areas was [(16
mm I.D.)2 / (4.6 mm I.D.)2] = 12.
Equation (1) could also be used for calculating the preparative injection
sample loading [29]. In this work, 0.075 mL sample with a concentration of
0.5 mg/mL was injected to the analytical column. So, a 1-mL sample with a
concentration of 6 mg/mL was loaded on preparative column. On the other
Recycling Preparative High Performance Liquid Chromatography 395

hand, according to equation 2, the flow rate was then increased to 12

mL/min for keeping the time frame for both small- and large-scale separa-
tions equivalent [29]. So, preparative HPLC separation was performed by
injecting 1 mL of acetone extract at room temperature with a constant flow-
rate of 12 mL/min. The identity and purity of the isolated compounds were
checked again by HPLC. As shown in Table IV, purity and resolution were
not critical, it was decided to use a somewhat lower flow rate than that pre-
dicted by equation 2 to gain some increase in resolution and purity. There-
fore, in constant ratio of methanol and water (50:50 v/v), the effect of flow
rate on purity of curcumin was studied. In this way, three flow rates (8, 10,
and 12 mL/min) were selected to study. As shown in Table IV, the best pu-
rity was achieved in 8 mL/min, but because of the increase in retention
time, 10 mL/min was selected as an optimizing flow rate. Then, the effect of
sample loading in five concentrations of acetone extract (0.5, 1, 2, 4, 6
mg/mL) on purity of curcumin was studied. As shown in Table V, much
pure curcumin was obtained in concentrations of 0.5 and 1 mg/mL at a con-
stant flow rate (10 mL/min) (Fig. 3C). However, when the amount of sam-
ple concentration was increased, poor resolution was achieved and caused
decrease of purity.

Fig. 4. Chromatogram of recycling preparative HPLC separation of curcuminoids by

using methanol and water as mobile phases with ratio of 50:50 (v/v), concentration of
1 mg/mL and flow rate of 10 mL/min
396 S. Mollayi et al.

Table IV. Purity percent of curcumin in preparative HPLC at various flow rates

Flow rate (mL/min) Retention Time (min) Purity (%)

8 21.3 97.91
10 12.5 95.32
12 10.1 93.43

Table V. Purity percent of curcumin in preparative HPLC at various concentrations and

constant flow rates (10 mL/min)

Concentration (mg/mL) Purity (%)

0.5 98.72
1 96.32
2 95.32
4 87.54
6 81.11

Equation 1:
2
⎡ (Columna l.D) ⎤ Columna leng h
Columna sampleload = Columnb sample load × ⎢ ⎥ × .
⎣ (Columnb l.D) ⎦ Columnb leng h
Equation 2:
2
⎡ (Columna l.D) ⎤
Columna sample load = Columnb sample load × ⎢ ⎥ .
⎣ (Columnb l.D) ⎦

Development of Recycling Preparative HPLC Method

According to the results which have been obtained from preparative HPLC,
achieving pure curcumin in high sample loading concentration by using
this technique was impossible. For obtaining this purpose, we used another
preparative technique called recycling preparative HPLC. In this technique,
curcuminoids were returned to the column after passing through the detec-
tor four times to achieve a higher purification (Fig. 4). At constant flow rate
and solvent mobile phases, the effect of sample loading in five concentra-
tions of acetone extract (0.5, 1, 2, 4, and 6 mg/mL) on purity of curcumin
was studied (Table VI). As shown in Table VI, up to 99.5% pure curcumin
was obtained by using this technique. Lastly, the solvent was dried by using
a freeze-dryer to obtain a purified compound.
Recycling Preparative High Performance Liquid Chromatography 397

Table VI. Purity percent of curcumin in recycling preparative HPLC at various

concentrations and constant flow rates (10 mL/min)

Concentration (mg/mL) Purity (%)

0.5 99.82
1 99.78
2 99.62
4 99.51
6 99.21

Conclusions
This study presents recycling preparative method for the purification of curcu-
min from curcuma longa L. Methanol and water (50:50 v/v) were selected as the
basic mobile phase system for recycling preparative HPLC. Curcumin was puri-
fied at a high purity of over 99.5% by this method. This method is simple,
highly efficient, environmentally friendly, and has been demonstrated to be ef-
fective for the preparation and purification of curcumin from curcuma longa L.

Acknowledgment
The authors gratefully acknowledge the Shahid Beheshti University, G.C.
for financial support of this study.

References
[1] B. Joe, M. Vijaykumar, and B.R. Lokesh, Crit. Rev. Food. Sci, 44, 97 (2004)
[2] F. Yang, G.P. Lim, A.N. Begum, O.J. Ubeda, and M.R. Simmons, J. Biol. Chem, 280,
5892 (2005)
[3] R. Wilken, M.S. Veena, M.B. Wang, and E.S. Srivatsan, Mol. Cancer, 10, 1 (2011)
[4] A. Shehzad, F. Wahid, and Y.S. Lee, Arch. Pharm, Chem. Life. Sci, 343, 489 (2010)
[5] H. Ahsan, N. Parveen, N.U. Khan, and S.M. Hadi, Chemico-Biol. Inter, 121, 161
(1999)
[6] D. Liu, J. Schwimer, Z. Liu, E.A. Woltering, and F.L. Greenway, Pharm. Biol, 46, 677
(2008)
[7] V.P. Kurup and C.S. Barrios, Mol. Nutr. Food. Res, 52, 1031 (2008)
[8] P. Basnet and N.S. Basnet, Molecules, 16, 4567 (2011)
[9] R.C. Reddy, P.G. Vatsala, V.G. Keshamouni, G. Padmanaban, and P.N. Rangarajan,
Biochem. Bioph. Res. Co, 326, 472 (2005)
398 S. Mollayi et al.

[10] P.R. Debata, M.R. Castellanos, J.E. Fata, S. Baggett, S. Rajupet, A. Szerszen, S. Be-
gum, A. Mata, V.V. Murty, L.M. Opitz, and P. Banerjee, Gynecol. Oncol, 129, 145
(2013)
[11] S. Barthelemy, L. Vergnes, M. Moynier, D. Guyot, S. Labidalle, and E. Bahraoui,
Res. Virology, 149, 43 (1998)
[12] A.P. Gupta, M.M. Gupta, and S. Kumar, J. Liq. Chrom. Rel. Technol, 22, 1561 (1999)
[13] S. Revathy, S. Elumalai, M. Benny, and B. Antony, J. Exp. Sci, 2, 21 (2011)
[14] P.S. Wakte, B.S. Sachin, A.A. Patil, D.M. Mohato, T.H. Band, and D.B. Shinde, Sep.
Purif. Technol, 79, 50 (2011)
[15] L. Xiang-zhou, Z. Yan-qiang, L. Yan-hua, and S. Chong-lu, Chem. Ind. Forest,
Prod, 26, 83 (2006)
[16] D.V. Dandekar and V.G. Gaikar, Separ. Sci. Technol, 37, 2669 (2002)
[17] V. Mandal, S. Dewanjee, R. Sahu, and S.C. Mandal, Nat. Prod. Commun, 4, 95
(2009)
[18] H.A. Aday, Eng. Tech. Journal, 29, 2087 (2011)
[19] J. Coll and Y. Tandron, Phytochem. Analysis, 16 (2005)
[20] O. Sticher, Nat. Prod. Rep, 25, 517 (2008)
[21] H.K. Teoh, E. Sorensen, and N. Titchener-Hooker, Chem. Eng. Sci, 58, 4145 (2003)
[22] S. Hellsten, J. Siitonen, M. Mänttäri, and T. Tuomo-Sainio, J. Chromatogr. A, 1251,
122 (2012)
[23] A.M. Anderson, M.S. Mitchell, and R.S. Mohan, J. Chem. Educ, 77, 359 (2000)
[24] K. Patel, G. Krishna, E. Sokoloski, and Y. Ito, J. Liq. Chrom. Rel. Technol, 23, 2209
(2000)
[25] K. Inoue, C. Nomura, S. Ito, A. Nagatsu, T. Hino, and H. Oka, J. Agric. Food. Chem,
56, 9328 (2008)
[26] A. Ghassempour, M. Ghahramanzamaneh, H. Hashempour, and K. Kargosha,
Acta. Chromatogr, 23, 641 (2011)
[27] H. Rezadoost and A. Ghassempour, Phytochem. Analysis, 23, 164 (2012)
[28] A. Rohman, Int. Food. Res. Journal, 19, 19 (2012)
[29] B.A. Bidlingmeyer, Preparative Liquid Chromatography, Elsevier, Amsterdam,
1987, pp. 1–104
Accepted by MWH