Analytical

Methods
View Article Online
PAPER View Journal | View Issue

Separation and identification of curcuminoids in

turmeric powder by HPLC using phenyl column
Published on 24 January 2014. Downloaded by Harvard University on 18/06/2014 22:08:30.

Cite this: Anal. Methods, 2014, 6, 2526

Imran Ali,* Ashanul Haque and Kishwar Saleem

Received 20th November 2013
Accepted 24th January 2014
DOI: 10.1039/c3ay41987h
www.rsc.org/methods
Natural products including curcuminoids are used globally for treating several diseases. Curcumin,
demethoxycurcumin and bis-demethoxycurcumin are the major constituents of Curcuma longa L. A
rapid, selective, efficient and reproducible HPLC method for the separation and identification of
curcuminoids was described using a Sunniest PhE (phenyl) column (250
4.6 mm, 5.0 mm). These
constituents were separated within 10.5 min using acetonitrile–methanol–water (40 : 20 : 40, v/v) as the
mobile phase with 1.0 mL min1 flow rate and 360 nm detection. The capacity factors (k, 4.2 to 4.9),
separation factors (a, 1.07 to 1.10) and resolution factors (Rs, 1.07 to 2.05) indicated a good separation of
the compounds. Attempts have also been made to describe the separation mechanism of the reported
method. The extraction of the curcuminoids from turmeric powder was 2.1, 0.46 and 0.1% for curcumin,
demethoxycurcumin and bis-demethoxycurcumin, respectively. The reported method was considered to
be novel due to base-line separation of curcuminoids, with sharp peaks and low LOD in comparison to
the reported methods in the literature. Briefly, the described method may be used for the quality control
of food stuffs and identification of curcuminoids in other natural products.

Commercially available turmeric powder is a mixture of

Introduction
The rhizomes of turmeric (Curcuma longa L.), a plant of the
Zingiberaceae family, provides a yellow, avoursome powder.
This yellow powder, known as turmeric, is mainly composed
of curcuminoid pigments along with resin and turmerone.
Turmeric has been used worldwide since ancient times as a
food ingredient due to its bright colour and promising health
properties.1 For a long time turmeric has been known for its
many medicinal values. It is used for curing sprains, swell-
ings, biliary disorders, rheumatism, sinusitis, abdominal
pains, icterus etc. In addition, turmeric has several other
important pharmaceutical properties such as anti-HIV, anti-
microbial, anti-oxidant, anti-parasitic and anti-cancer
activity.2–10 These activities of curcuminoids are mainly due
to the presence of three structurally correlated curcuminoids,
viz. 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-
dione (curcumin, C), 1-(4-hydroxyphenyl)-7-(4-hydroxy-3-
methoxyphenyl)-1,6-heptadiene-3,5-dione (demethoxycur-
cumin, DMC) and 1,7-bis(4-hydroxyphenyl)-1,6-heptadiene-
3,5-dione(bis-demethoxycurcumin, BDMC).10 The anticancer
activities order of these curcuminoids is BDMC > DMC > C.11
The structures of these curcuminoids are given in Fig. 1,
showing two substituted phenyl rings separated by hepta-
dienone spacer.
Department of Chemistry, Jamia Millia Islamia (Central University), Jamia Nagar, New
Delhi – 110025, India. E-mail: drimran_ali@yahoo.com; drimran.chiral@gmail.com;
Web: http://jmi.ac.in/iali2; Tel: +91-9211458226
naturally occurring curcuminoids with curcumin (C) as the
major constituent and the other two (DMC and BDMC) as minor
components (C : DMC : BDMC ¼ 77 : 18 : 5%).2,12 Due to the
different pharmaceutical properties of the three curcuminoids,
their separation and identication are important issues in
medicinal chemistry. A thorough search of the literature was
carried out and some methods for analysis of curcuminoids are
available. The reported methods include TLC,12–17 column
chromatography,12,18 HPTLC,19 and HPLC.12,20–40 A comparison
of the reported methods was carried out with the observation
that TLC and HPTLC are time consuming methods with poor
limits of detection. In all cases C18, amine and core shell
(accucore) columns were used in HPLC, which were able to
separate three curcuminoids but with some limitations. The
major limitations include extremely low pH of the mobile
phases and high separation time and detection limits. In some
papers, the peaks were very close to one another, while peaks
were broad in some others. This may be due to the poor inter-
action of these molecules with reverse phase materials because
of the absence of p–p interactions required for such kinds of
molecules due the presence of phenyl moieties. Therefore, the
reported methods showed poor separation and broad peaks
with high limits of detection and quantication. In view of these
facts, attempts have been made to develop, optimize and
validate a fast, selective and reproducible method for the
separation and identication of a mixture of curcuminoids
using a phenyl HPLC column. The developed, optimized and
validated method was applied for the analysis of these three

2526 | Anal. Methods, 2014, 6, 2526–2536 This journal is © The Royal Society of Chemistry 2014

Paper
Published on 24 January 2014. Downloaded by Harvard University on 18/06/2014 22:08:30.
Fig. 1 Structures of curcuminoids.
curcuminoids in commercial turmeric powder. The results of
these investigations are reported herein.
Experimental
Chemicals and reagents
Turmeric powder was purchased from a local market in New
Delhi, India. The standard samples of the curcuminoids were
kindly supplied by SAMI Labs, Bangalore. Acetonitrile and
methanol of HPLC grade were purchased from Fisher Scientic,
Fairlawn, New Jersey, USA. Puried water was prepared by using
a Millipore Milli-Q, Bedford, USA water purication system. A
UV spectrometer by PG instruments (model T80) was used.
Preparation of standard solutions
The standard solutions (1.0 mg mL1) of each curcuminoid and
their mixture in commercial samples were prepared in aceto-
nitrile. The stock solutions were protected from light by
covering with aluminium foil and stored at 4  C. A grade bulb
pipettes and 10.0 mL volumetric asks were used for serial
dilutions of these curcuminoids with acetonitrile to obtain the
required concentration ranges (0.01–0.10 mg mL1).
Extraction of curcuminoids from turmeric powder
Curcuminoids are soluble in various organic solvents including
ethanol, dichloromethane and ethyl acetate. Some research
papers describe the application of various solvents for extraction
of curcuminoids from turmeric powder.41–43 In addition,
sequential extraction with a number of solvents has also been
used for this purpose.44,45 It was observed that DCM is the best
solvent for the extraction of curcuminoids from turmeric powder.
Therefore, DCM was used as extracting solvent as per the
procedure described by Anderson et al.42 50.0 g of turmeric
powder was taken in a 250 mL round bottom ask and 125.0 mL
dichloromethane was added, followed by constant stirring with a
magnetic stirrer. The mixture was reuxed for 1.0 h at 50  C. It
was ltered by a Buchner funnel, followed by the separation of
the mother liquor. The mother liquor was concentrated on a
rotary evaporator, resulting in a dark orange oily liquid, which
This journal is © The Royal Society of Chemistry 2014
View Article Online
Analytical Methods
was precipitated with the addition of 50.0 mL hexane. The
precipitate was further ltered through a Buchner funnel, which
gave a mixture of curcuminoids. The purity of the extracted
curcuminoids was conrmed by a UV-vis. spectrophotometer.
The resulted product was analysed by HPLC for curcuminoids.
HPLC instrument
The HPLC system used was by ECOM (Prague, Czech Republic),
consisting of a solvent delivery pump (model Alpha 10), manual
injector, absorbance detector (Sapphire 600 UV-vis.), chroma-
tography I/F module data integrator (Indtech. Instrument,
Mumbai, India) and Winchrome soware. The column used
was a Sunniest PhE (phenyl ethyl) column (250
4.6 mm,
5.0 mm) by Chromanik, Japan.
HPLC conditions
All the experiments were carried out using the HPLC system
described above. Aliquots of 5.0 mL of standard solutions of
each curcuminoid and their mixture in a commercial sample
(1.0 mg mL1 in acetonitrile) were loaded onto the HPLC
instrument, separately and respectively. The mobile phase used
was acetonitrile–methanol–water (40 : 20 : 40, v/v) in isocratic
mode (1.0 mL min1). The mobile phase was prepared, ltered
and degassed daily before use. All the experiments were carried
out at 27  1  C with detection at 360 nm. The chromatographic
parameters such as retention (k), separation (a) and resolution
(Rs) factors were calculated. The order of elution was ascer-
tained by running individual curcuminoids. The qualitative and
quantitative analyses were carried out by using retention times
and peak areas, respectively. The chromatographic method was
optimized and validated by carrying out extensive experimen-
tation, followed by applied analysis of curcuminoids in the
extracted turmeric powder.
Validation
The validation of the HPLC method was carried out by calcu-
lating various HPLC parameters. The linearity, limit of detection
Anal. Methods, 2014, 6, 2526–2536 | 2527

Analytical Methods
(LOD), limit of quantitation (LOQ), precision, accuracy, selec-
tivity, robustness and ruggedness were determined for the
purpose. The LOD and LOQ were determined by injecting more
diluted samples of curcuminoids. The results of the statistical
analyses of the experimental data, such as relative standard
deviation, correlation coefficients and condence limit, were
calculated using Microso Excel. Good linearity of the calibra-
tion graphs and the negligible scatter of experimental points
were considered for calculations of correlation coefficients and
relative standard deviations.46 The robustness of the method was
Published on 24 January 2014. Downloaded by Harvard University on 18/06/2014 22:08:30.
demonstrated by the versatility of the experimental factors that
affected the peak areas.
Linearity
The linearity was tested by least squares linear regression
analysis of the calibration curve.46 The linearity of calibration
curves (peak area vs. concentration) for curcuminoids standards
as well as in turmeric powder was checked over the concentra-
tion range of 0.01.0–0.10 mg mL1. Equal volumes (5.0 mL) of
the standards as described above were loaded onto the HPLC
instrument. The chromatograms were recorded, separately and
respectively. The calibration curves of all curcuminoids were
constructed, separately and respectively, using the observed
peak areas versus nominal concentrations of the analytes.
Detection and quantitation limits
The LOD and LOQ were determined as three and ve times the
baseline noise, respectively, following the United States
Pharmacopoeia.46
Specicity
The specicity of the method was investigated by observing any
interference in chromatographic parameters due to the present
of some impurities in standard samples. The standard samples
were mixed with small amount of turmeric powder to make
them impure.
Precision
To calculate precision data, three different concentrations (0.01,
0.05 and 0.10 mg mL1) of each curcuminoids were used. Five
sets of the chromatographic runs were carried out for each of
the three concentrations.
Accuracy
Different concentrations of curcuminoids were used to deter-
mine the accuracy of the HPLC method. Three concentrations
i.e. 0.01, 0.05 and 0.10 mg mL1 were used. The chromato-
graphic runs were carried out ve times (n ¼ 5). The accuracy
was determined by interpolation of ve replicates peak areas of
these molecules.
Robustness
The method robustness was determined by a slight variation in
the chromatographic parameters such as ow rate, tempera-
ture, mobile phase composition and wavelength. The retention
2528 | Anal. Methods, 2014, 6, 2526–2536
View Article Online
Paper
time, peak area and shape were analyzed under the established
and slightly varied experimental conditions.
Ruggedness
The ruggedness of the method was determined by changes in
the experimental environment, such as different operators and
different days.
Results and discussion
Extraction of curcuminoids from turmeric powder
As discussed above, 1.40 g of curcuminoids were obtained from
50.0 g turmeric powder. The amounts of curcumin, demethoxy-
curcumin and bis-demethoxycurcumin were 1.06, 0.23 and 0.05
g, respectively (Table 1). Fig. 2 depicts the UV-vis. spectra of the
curcuminoids in methanol (2
105 M) (Fig. 2). lmax. values of
C, DMC and BDMC were 420, 416 and 412 nm, respectively.
The percentage recoveries of the compounds were 2.1, 0.46
and 0.1 for C, DMC and BDMC, respectively. The values of RSD,
correlation coefficient and condence level were 2.0–2.5, 0.9998–
0.9999 and 98.80–99.00, respectively (Table 1). Attempts have
been made to extract these curcuminoids from turmeric powder
by using methanol, acetonitrile, ethyl acetate and ethanol as
pure solvents or as their mixtures but maximum recoveries could
be achieved with pure dichloromethane only. Therefore,
dichloromethane was used as the extracting solvent for the
reported curcuminoids. Attempts have been made to compare
the extraction recoveries with those reported in the literature.41–45
It was observed that the extraction recoveries of these curcumi-
noids were comparable. The extracted curcuminoids were pure
as there was no extra peaks present in the HPLC studies.
HPLC method development
The separation and identication of curcuminoids were carried
out using the column and mobile phase as described in the HPLC
instrumentation section. The separated curcuminoids from
commercial samples were identied by running and comparing
the retention times of the individual curcuminoids. Calibration
curves were plotted for all curcuminoids and used to determine
their concentrations in turmeric powder. Quantitative analysis of
the curcuminoids in turmeric powder was carried out by
comparing their peak areas with those of the standards. The
capacity (k), separation (a) and resolution (Rs) factors for these
compounds in standard solutions and turmeric powder were
calculated and are given in Table 2. The chromatograms of the
curcuminoid mixtures in standard and extracted samples are
given in Fig. 3. It is clear from this gure that all three curcu-
minoids are base-line separated with sharp peaks within 10.5
min. The order of elution observed was BDMC, DMC, C. The
capacity, separation and resolution factors of the curcuminoids
were 4.2 to 4.9, 1.07 to 1.10 and 1.07 to 2.05, respectively (Table 2).
HPLC method optimization
To optimize the chromatographic conditions, various combina-
tions of acetonitrile–methanol–water were tried. The inuence of
This journal is © The Royal Society of Chemistry 2014

Paper
Table 1 Regression analyses data for the extraction of curcuminoids from turmeric powdera
Extraction
Curcuminoids Recovery (%) % RSD
Curcumin 2.1 2.50
Demethoxycurcumin 0.46 2.40
Bis-demethoxycurcumin 0.1 2.00
a
n ¼ 5.
Published on 24 January 2014. Downloaded by Harvard University on 18/06/2014 22:08:30.
ow, pH, detection wavelength and volume injected were
studied. Other solvents such as phosphate buffer, acetate buffer
and ratios of different organic solvents were also tested. As a
result of exhaustive experimentation, the best chromatographic
conditions were optimized and are reported herein. The opti-
mization of chromatographic parameters is discussed in the
following paragraphs.
Effect of pH
The pH of the mobile phase is one of the important factors in
controlling HPLC separation. The pH of the mobile phase was
varied from 3.0–7.0. Above pH 7.0, the probability of curcumi-
noid decomposition is high,32 which is why the experiments
were carried out up to a maximum pH of 7.0. The resolution
factors ranged from 0.88 to 1.32 for the molecules at pH 3.0–6.0,
while the values at pH 7.0 were 1.07 to 1.45 (Fig. 4). Good
separation of the reported molecules at pH 7.0 might be due to
the presence of curcuminoid phenoxide ions (having higher p
electron density), leading to p–p interactions with the phenyl
column. Contrarily, this situation is not available at low pH
(in acidic media) and, hence, results in poor separation.
Effect of acetonitrile
The amount of acetonitrile was varied from 10 to 60 parts,
keeping methanol and water constant. Interestingly, it was
observed that there were no peaks within 20 min with 10 mL
acetonitrile (low amount). Upon further increasing the volume
of acetonitrile from 10 / 20, two peaks (10.5 and 11.1 min)
were detected. Furthermore, at 30 parts of acetonitrile, three
clear peaks (14.6, 15.5 and 16.6 min) were observed, with Rs
values of 0.95, 0.71 and 0.78 for C, DMC and BDMC,
Fig. 2 UV-vis. spectra of curcuminoids in methanol (2
105 M).
This journal is © The Royal Society of Chemistry 2014
View Article Online
Analytical Methods
Correlation coefficient (R2) Condence level (%)
0.9998 98.78
0.9999 98.80
0.9999 99.00
respectively. However, this system had the drawback of low
detection of DMC and BDMC. Upon a further increment to
40 mL, better peak shape with clear separation was observed,
with Rs values of 1.27, 1.07 and 2.05 for C, DMC and BDMC,
respectively. These values indicated satisfactory separation.
Further increase in acetonitrile concentration to 50 and 60 mL
resulted in poorer resolution, which might be due to weak
bonding between molecules and the column stationary phase
due to the effects of acetonitrile (p-electrons of the nitrile
functionality involved in bonding with phenyl group by
replacing analyte molecules).47 As a result, the best volume of
acetonitrile was selected as 40 mL (Fig. 5).
Effect of methanol
The amount of methanol was varied from 10 to 60 mL. The
resolution was poor (Rs ¼ 1.15, 0.85 and 1.7) at a low amount of
methanol (10 mL). However, at 20 mL of methanol Rs values
were slightly higher i.e. 1.5, 1.07 and 1.56. With further increase
of methanol, Rs values decreased with broad peaks, which
might be attributed to the decreasing concentration of aceto-
nitrile, responsible for sharp peaks. The results of this optimi-
zation are shown in Fig. 6.
Effect of water
The amount of water was varied from 10 to 60 mL. Only two
peaks (3.73 and 4.32 min) were observed with 10 mL water in the
mobile phase, which might be attributed to the high polarity of
the solvent. Upon further increasing the water content (20 / 40),
three peaks were observed within 10.5 min with poor resolution
at 30 mL. However, peaks were well resolved and sharp with
40 mL of water. Further increase in the water content resulted in
Anal. Methods, 2014, 6, 2526–2536 | 2529
 View Article Online

Analytical Methods Paper

Table 2 Chromatographic and precision data of curcuminoidsa,b,c,d

% Correlation Condence
Curcuminoid k a Rs RSD coefficient level (%)

Standard solutions
Curcumin 4.28 1.07 1.27 2.00 0.9999 99.00
Demethoxycurcumin 4.59 — — — — —
Bis-demethoxycurcumin 4.92 1.10 1.07 2.05 0.9999 99.00

Turmeric powder extract

Curcumin 4.23 1.05 1.06 2.00 0.9999 98.80
— — — — —
Published on 24 January 2014. Downloaded by Harvard University on 18/06/2014 22:08:30.

Demethoxycurcumin 4.50
Bis-demethoxycurcumin 4.86 1.08 1.06 2.10 0.9999 99.00
a
n ¼ 5. b Note: k: capacity factor, a: separation factor and Rs: resolution factor. c Column: phenyl column (250
4.6 mm, 5.0 mm). d Isocratic HPLC
conditions: acetonitrile–methanol–water (40 : 20 : 40, v/v), ow rate: 1.0 mL min1, UV detection: 360 nm, temperature: 27  1  C.

partial separation with broad peaks. Therefore, 40 mL water gave

the best results.

Effect of ow rate

The ow rate of the solvent system acetonitrile–methanol–
water (40 : 20 : 40, v/v) was varied from 0.5 to 2.0 mL min1
and the chromatograms are shown in Fig. 7. It was observed
that at low ow rate (0.5 mL min1), the peaks were poorly
resolved with high retention time (Fig. 7). An increase in the
ow rate to 1.0 mL min1 resulted in sharp base-line separa-
tion. On the other hand, high ow rates (1.5 and 2.0 mL
min1) decreased retention times with partial separation.
Fig. 8 depicts the typical trend of Rs change with respect to
ow rate. At 0.5 mL min1, the values of Rs were 0.79, 0.95 and Fig. 4 Effect of pH on the resolution of curcuminoids. (Rs1: resolu-
1.25 for C, DMC and BDMC, respectively. Upon increasing the tion between peak 1–2, Rs2: resolution between peak 2–3 and Rs3:
resolution between peak 1–3. Peak 1: BDMC, peak 2: DMC and peak
ow rate to 1.0 mL min1, there was an increase in these
3: C).
values (Rs ¼ 1.06, 1.26 and 1.6). At high ow rates (1.5 and
2.0 mL min1), there was decrease in resolution among the
peaks (Rs ¼ 0.92, 1.18 and 1.3). Briey, the peaks were well
resolved at 1.0 mL min1 ow rate, which was considered as acid free conditions and has good efficiency. It is well known
the best ow rate. that van der Waal's forces, hydrogen bondings, dipole-induced
dipole interactions, ionic interactions and coordination
bonding are separation controlling forces in HPLC. It is inter-
Mechanism of separation esting to note that the reported curcuminoids have little
As mentioned in the introduction, curcuminoids were poorly difference in their structures and such types of forces are almost
resolved on C18 and amine columns but the present method is of equal magnitudes. Hence, C18 and amine phases involving
more advantageous as it works at normal temperature, under the above-mentioned interactions are not capable of separating

Fig. 3 HPLC chromatograms of (A) the standard mixture and (B) extracted curcuminoids. Experimental conditions are as given in the text.

2530 | Anal. Methods, 2014, 6, 2526–2536 This journal is © The Royal Society of Chemistry 2014

Paper
Published on 24 January 2014. Downloaded by Harvard University on 18/06/2014 22:08:30.
Fig. 5 Effect of acetonitrile on the resolution of peaks. (Rs1: resolution
between peak 1–2, Rs2: resolution between peak 2–3 and Rs3: reso-
lution between peak 1–3). (Peak 1: BDMC, peak 2: DMC and peak 3: C).
Fig. 6 Effect of methanol on the resolution of peaks. (Rs1: resolution
between peak 1–2, Rs2: resolution between peak 2–3 and Rs3: reso-
lution between peak 1–3. Peak 1: BDMC, peak 2: DMC and peak 3: C).
Fig. 7 Effect of flow rates on the retention times of the curcuminoids.
them successfully. It is important to mention here that p–p
interactions, cation–p or anion–p interactions have greater
strength than the bondings discussed above. Due to high
strength of these interactions the reported molecules are
differentiated by the stationary phase (phenyl ethyl silica);
This journal is © The Royal Society of Chemistry 2014
View Article Online
Analytical Methods
Fig. 8 Effect of flow rate on the resolution of peaks. (Rs1: resolution
between peak 1–2, Rs2: resolution between peak 2–3 and Rs3: reso-
lution between peak 1–3. Peak 1: BDMC, peak 2: DMC and peak 3: C).
resulting into their good separation. Fig. 1 clearly indicates the
presence of two phenyl moieties in these molecules, on each
side. Therefore, the phenyl column was used to overcome the
problem of their poor separation. For these molecules, p–p-
interactions played a major role in their separation. Addition-
ally, the above-mentioned forces also contribute in separation
mechanism. The phenyl column comprises several Si–O–
(CH2)2–Ph groups (Fig. 9). The phenyl groups on the stationary
phase retained the reported molecules to different extents,
which might be due to dissimilar magnitudes of p–p-interac-
tions between the curcuminoids and stationary phase, resulting
in good resolution. Besides, due to the comparatively stronger
p–p-interactions, the diffusion of curcuminoids is reduced,
resulting in big and sharp peaks (low detection limits). Briey,
the separation of the reported curcuminoids is controlled by
p–p interactions along with other forces.
The phenyl ethyl column (alkyl chain stationary phase
(C8/C18) is replaced by aromatic rings) has good potential for the
analyses of natural products and other pharmaceuticals by
exploiting the p–p type interactions with analytes.48 On the
reported column, the p–p-reversed-phase (p-RP) retention
mechanism is found to be prominent,49 with some other addi-
tional interactions.50 Even stereoisomers with identical prop-
erties can be separated by exploiting p–p type interactions on
chiral columns.47,51–53 It has already been established that
solutes with p-electron systems display different retention
behaviors on p-RP-phases than on ordinary RP-columns.
Contrarily, solutes without p-electrons or with sterically
hindered p-electron systems behaved in similar fashions on
C8/C18 and p-RP-phases.54 Stronger p–p interactions are
responsible for higher aromatic solute retention on p-RP-pha-
ses.55,56 As per Euerby et al.,57 selectivity of the phenyl ring
bonded stationary phase varied with the length of the spacer
between the phenyl group and the silica surface. The authors
reported low selectivity on columns having the phenyl ring
directly attached to the silica. However, selectivity of the
stationary phase is augmented as the number of linker atoms
between phenyl and silica increases. The authors observed poor
p–p interactions on a column with the phenyl ring close to
the silica surface due to p–p stacking among analytes and
Anal. Methods, 2014, 6, 2526–2536 | 2531

Analytical Methods
Published on 24 January 2014. Downloaded by Harvard University on 18/06/2014 22:08:30.
Fig. 9 Possible p–p interactions between the stationary phase (phenyl ethyl phase) and curcuminoids (C, DMC and BDMC). Solid and dotted
lines represent strong and weak interactions, respectively.
stationary phases. On introduction of linker atoms, the p–p
interactions among analytes and stationary phase were suffi-
cient, resulting in good separation. These results indicated the
phenyl ethyl column as the best choice for the separation of
analyses with p electrons.
Attempts have been made to develop the mechanism of
separation at a supra-molecular level (Fig. 9). Curcuminoid
structures can be differentiated from one another by the pres-
ence or absence of methoxy groups (an electron donating
group). The greater the number of methoxy groups, the greater
the electron density on the aromatic ring and, hence, the greater
the p–p interactions. The experiments were carried out at pH
7.0 and all of the curcuminoids occur in the phenoxide ionic
form at this pH, increasing p electron density on the phenyl
rings. Therefore, there are stronger p–p interactions at pH 7.0
in comparison to acidic pH. The methoxy group affects p elec-
tron density on the phenyl rings of these molecules. C and DMC
have two methoxy groups while BDMC has none, which means
that p electron density on these molecules is in the order of C >
DMC > BDMC. Similarly, the binding capacity of these mole-
cules on stationary phase will be in the same order. Conse-
quently, the elution order of these compounds will be reversed,
which was observed experimentally. Therefore, it may be
assumed that the separation of curcuminoids on a phenyl phase
is being controlled by p–p interactions along with other forces.
Validation of HPLC method
The HPLC method was validated with respect to various
parameters including linearity, limit of detection (LOD), limit of
quantitation (LOQ), precision, accuracy, selectivity, robustness
and ruggedness.58
2532 | Anal. Methods, 2014, 6, 2526–2536
View Article Online
Paper
Linearity
The linearity of the calibration curves (peak area vs. concen-
tration) for the curcuminoid standards as well as for turmeric
powder were checked over the concentration ranges of 0.01–
0.1 mg mL1. The plotted curves were linear over these
concentration ranges (n ¼ 5) for all curcuminoids. The peak
areas of curcuminoids were plotted versus their respective
concentrations. The linear regression analysis was performed
on the resultant curves. The correlation coefficient (R2) (n ¼ 5)
was found to be 0.9999 for all curcuminoids. The values of RSD
and condence levels were in the range of 2.00–2.10 and 98.80–
99.00, respectively, across the concentration ranges studied.
Detection and quantitation limits
The values for LOD and LOQ for curcuminoids ranged from
0.30–0.50 ng and 1.00–2.00 ng, respectively. The resultant RSDs
for these studies were in the range of 2.00–2.50%.
Specicity
The method has quite good specicity, as can be seen from
Fig. 3. The retention times of all curcuminoids are almost the
same in both standard solutions and turmeric powder. There
was no effect from added impurities in standards on the
retention times and peak shape of these curcuminoids. These
ndings indicate a good specicity of the reported method.
Precision
The precision data was calculated by taking three concentra-
tions of all curcuminoids i.e. 0.01, 0.05 and 0.10 mg mL1. Five
chromatographic runs were carried out for all the three
This journal is © The Royal Society of Chemistry 2014
 View Article Online

Paper Analytical Methods

Table 3 A comparison of curcuminoids separation by HPLC

Separation Detection limit &

No. Column Mobile phase time (min) ow rate Drawbacks Ref.

1. C18 CH3OH–2% CH3COOH– 6.75 —, 1 mL min1 Very low difference in 12

CH3CN retention time; low pH
leading to column damage;
detection limits not given
2. C18 (A) H2O (0.25% 14.0 —, 0.2 mL min1 Carried out at 48  C; low pH 20
CH3COOH) leading to column damage;
and (B) CH3CN, detection limits not given
Published on 24 January 2014. Downloaded by Harvard University on 18/06/2014 22:08:30.

0–17 min, 40–

60% B; 17–32 min,
60–100%
B; 32–38 min, 100%
B; 38–40
min, 100–40% B
3. RP-5-NH2 C2H5OH–H2O 20.73 —, 1 mL min1 High separation time and, 23
(96 : 04, v/v) hence costly; amino-bonded
stationary phase has a catalytic
effect upon curcumin degradation;
compositional variations lead to
variable results (due to the use of
azeotropic mixture of ethanol);
detection limits not given
4. C18 0.1 M of acetate buffer 28 1.5, 0.9 and High retention time and, hence, 24
(pH 4.0)–CH3CN 0.09 ng mL1 for C, costly; low pH leading to damage
(57 : 43, v/v) DMC & BDMC, of column
1 mL min1
5. C18 Gradient elution 11 —, 1 mL min1 Low pH leading to column damage; 27
mobile phase solvents detection limits not given
(A) 2% CH3COOH in
H2O and (B) CH3CN
6. C18 CH3CN–CH3OH–H2O 12 100–5000 ng mL1, Low pH leading to damage 29
(40 : 23 : 37, v/v) pH 3.0 1 mL min1 of column; high detection limits
7. C18 Gradient elution mobile 13.2 —, 0.7 mL min1 Retention times too close; low pH 30
phase solvents (A) 3 mM leading to column damage; detection
H3PO4 in H2O and (B) CH3CN limits not given
8. C18 0.05% CH3COOH–CH3OH 26 —, 1 mL min1 High retention time and, hence, 31
(15 : 85, v/v) costly; low pH leading to column
damage; detection limits not given
9. C18 Gradient elution mobile 17.0 —, 1 mL min1 High retention time and, hence, 32
phase solvents (A) 0.25% costly; low pH leading to column
CH3COOH in H2O and damage; detection limits not given
(B) CH3CN
10. C18 CH3CN–2% CH3COOH 13.6 0.90, 0.84 and Low pH leading to damage of 33
(40 : 60, v/v) 0.08 mg/ column; high detection limits
for C, DMC &
BDMC,
2 mL min1
11. C18 CH3CN–0.1% CF3COOH– 9.0 27.99, 31.91 and Low pH leading to damage of 34
(50 : 50, v/v), (pH adjusted to 21.81 ng mL1 for column; moderate detection limits
3.0 with NH3) C, DMC & BDMC,
1.5 mL min1
12. TSK-GEL 0.1% HCOOH–CH3CN 10.0 —, 1.0 mL min1 Low pH leading to damage of 35
ODS 80 Ts (50 : 50, v/v) column; detection limits not given
13. C18 40% THF–60% H2O 9.27 —, 0.7 mL min1 Low pH leading to damage of 36
with 1% citric acid, pH 3.0 column; broad peaks, detection
limits not given
14. C18 CH3CN–5% CH3COOH 2.0 —, 1.0 mL min1 Extremely low pH leading to damage 37
(75 : 25, v/v) of column; detection limits not given
15. C18 1% H3PO4–CH3CN 6.36 2.5 mg mL1, Low pH leading to damage of column; 38
1.0 mL min1 detection limit high
16. Chromolith H2O–CH3CN–CH3COOH 20.5 50 ng mL1, High retention time and, hence, 39
column (60 : 40 : 1, v/v) 1.0 mL min1 costly; retention times too close
(monolithic C18)

This journal is © The Royal Society of Chemistry 2014 Anal. Methods, 2014, 6, 2526–2536 | 2533
 View Article Online

Analytical Methods Paper

Table 3 (Contd. )

Separation Detection limit &

No. Column Mobile phase time (min) ow rate Drawbacks Ref.

17. C18 5.0 mM CH3CN–H3PO4 18.6 0.207, 0.202 and High retention time 40
(45 : 55, v/v) 0.514 ng mL1 for and, hence, costly;
CUR, DMC and detection limit high
BDMC, 1.0 mL min1
18. C18 and MeOH–10 mM H3PO4 3.0 —, 0.8 mL min1 Not capable of separating 59
accucore (80 : 20, v/v) under normal conditions
(room temperature) and
Published on 24 January 2014. Downloaded by Harvard University on 18/06/2014 22:08:30.

low pH leading to damage

of column
19. RP-phenyl ACN–MeOH–H2O 10.5 0.30–0.50 ng, Fast, reproducible, at a Present
column (40 : 20 : 40, v/v) 1.0 mL min1 new wavelength work

concentrations. RSDs were calculated and ranged from 2.00 to
2.80%;, indicating the HPLC method to be precise.
Accuracy
The accuracy of the method was tested by analyzing different
samples of the curcuminoids as described in the Experimental
section. The accuracy was determined by interpolation of
replicate (n ¼ 5) peak areas of three accuracy standards. In each
case, the percent error was calculated and found in the range of
0.60 to 1.20%. This range indicated a good accuracy of the
developed method.
Robustness
Small changes were made including in mobile phase compo-
sitions, ow rates, amounts loaded and detection wavelengths.
It was observed that there were no remarkable variations in the
HPLC results. No change in the HPLC results upon varying the
above experimental conditions indicated the reported method
to be robust.
Ruggedness
The ruggedness assessment was performed during the devel-
opment of the HPLC method. RSD values for intra- and inter-
days of curcuminoids were in the range of 2.00–2.51, indicating
the robustness of the method. Besides, the results obtained
with different operators were unaffected, which also indicated
ruggedness of the method.
Comparison with other methods
The reported results of the curcuminoid separation were
compared with those reported in the literature.12,20,23,24,27,29–40,59
The comparison was carried out in terms of columns, mobile
phase, ow rates, detection limits, peak shapes and economy.
The data is given in Table 3. It is clear from this table that all the
methods used reversed phase C18, accucore and amine columns
with acid in the mobile phases. The pH of these mobile phases
are expected to be between 1 and 2, which damages the HPLC
column. Only one column is based on an amine group, which
has a catalytic effect upon curcuminoid degradation and
compositional variations, leading to variable results. Most
methods used 1.5–2.0 mL min1 as ow rates, using moderate
amounts of costly HPLC grade solvents. Besides, the retention
times of these methods ranged over 11–28 min. In this way,
these methods are costly and time consuming. Additionally, in
most of the cases the detection limits are not given as the
separation is poor on C18 columns. In some cases the detection
limits are given but these are high and not acceptable. In some
papers the peaks are not well resolved and sharp. Of course, the
separation time is 3.0 min on an accucore column but this
column is not capable of separating curcuminoids under
natural laboratory conditions. The HPLC instrument needs a
heating device to achieve 40  C for good separation. Besides, the
low pH damaging the column was another major drawback of
the accucore column. On the other hand, the reported method
on the phenyl based reversed phase column showed sharp
peaks with good resolution factors. The separation time is only
10.5 min with low limits of detection i.e. 0.30–0.50 ng. The pH of
the mobile phase is 7.0, which did not affect the column life.
Short experimental time and use of water made this method
economic and eco-friendly. By considering all these factors, it
was concluded that the presented HPLC method was superior to
the reported ones in terms of economy and eco-friendliness,
with low limits of detection and sharp peaks.
Application of developed and validated
HPLC method
The validity of the developed method was applied to analyze cur-
cuminoids in turmeric powder extract. The qualitative and quan-
titative analyses of curcuminoids were carried out by using the
above-mentioned HPLC conditions. The chromatograms of cur-
cuminoids in turmeric powder are shown in Fig. 3. The quantita-
tive analysis of curcuminoids in turmeric powder was carried out
by comparing their peak areas with those of standards. For
calculation of concentrations of curcuminoids in turmeric powder
extract, ve sets of HPLC experiments were carried out under
identical experimental conditions. The amounts of curcumin,

2534 | Anal. Methods, 2014, 6, 2526–2536 This journal is © The Royal Society of Chemistry 2014

Paper
demethoxycurcumin and bis-demethoxycurcumin in turmeric
powder were 21.2, 4.60 and 1.0 g kg1, respectively.
10
Conclusion 11
A fast, reproducible, selective and effective HPLC method on 12
phenyl reversed phase column was described for the analysis of
curcuminoids within 10.5 minutes at 360 nm. The values of the 13
LOD and LOQ for the curcuminoids ranged from 0.30–0.50 ng
and 1.00–2.00 ng, respectively. Linearity was observed in the 14
Published on 24 January 2014. Downloaded by Harvard University on 18/06/2014 22:08:30.
concentration range of 0.01 to 0.10 mg mL1 for all of the cur-
cuminoids. The method was used for analysis of curcuminoids 15
in turmeric powder with concentrations of curcumin, deme-
thoxycurcumin and bis-demethoxycurcumin determined as 16
21.2, 4.60 and 1.0 g kg1, respectively. The results of the
proposed method were compared (Table 3) and it was observed 17
that the present method had advantages such as working at
normal temperature, using acid free conditions and having
good efficiency. Besides, the reported method can be used for 18
the quality control of any food stuff containing turmeric.
Besides, the reported method can be used to identify these 19
curcuminoids in food stuffs and other natural products. The
base-line separation, with good values of separation and reso- 20
lution factors, of these molecules on a phenyl column dictated
its application at the preparative scale for the separation of 21
individual curcuminoids.
22
Acknowledgements
23
The authors are thankful to CSIR (Council of Scientic and
Industrial Research), New Delhi for providing a Senior Research 24
Fellowship to Mr Ashanul Haque. The authors also thanks Dr T.
Samuel Manoharan, Principal Scientist & GM (R&D), SAMI Labs, 25
Bangalore for supplying curcuminoids as gi sample for the
research work. Thank is also to Chromanik, Japan for providing 26
the phenyl column as a gi.
27
References
28
1 H. P. T. Ammon and M. A. Wahl, Planta Med., 1991, 57, 1–7.
2 H. Ahsan, N. Parveen, N. U. Khan and S. M. Hadi, Chem.-Biol.
Interact., 1999, 121, 161–175. 29
3 D. S. H. L. Kim, S. Y. Park and J. Y. Kim, Neurosci. Lett., 2001,
303, 57–61. 30
4 M. D. Mesa, M. C. Ramirez-Tortosa, C. M. Aguilera,
A. Ramirez-Bosca and A. Gil, Ars Pharm., 2000, 141, 307–321. 31
5 G. K. Reddy, G. Chandrakasan and S. C. Dhar, Biochem.
Pharmacol., 1989, 38, 3527–3534.
6 A. Simon, D. P. Allais, J. L. Duroux, J. P. Basly, S. Durand- 32
Fontainer and C. Delage, Cancer Lett., 1998, 29, 111–116.
7 I. Ali, A. Haque, K. Saleem and M. F. Hsieh, Bioorg. Med. 33
Chem., 2013, 21, 3808–3820.
8 I. Ali, K. Saleem, D. Wesselinova and A. Haque, Med. Chem. 34
Res., 2013, 22, 1386–1398.
9 Turmeric: The genus Curcuma. Medicinal and Aromatic Plants- 35
Industrial Proles. ed. P. N. Ravindran, K. N. Babu and
This journal is © The Royal Society of Chemistry 2014
View Article Online
Analytical Methods
K. Sivaraman, CRC Press, Taylor & Francis Group, Boca
Raton, London, 2007.
A. J. Ruby, G. Kuttan, K. D. Babu, K. N. Rajasekharan and
R. Kuttan, Cancer Lett., 1995, 94, 79–83.
P. H. Bong, Bull. Korean Chem. Soc., 2000, 21, 81–86.
G. K. Jayaprakasha, L. J. M. Rao and K. S. Sakariah, J. Agric.
Food Chem., 2002, 50, 3668–3672.
H. B. Ramussen, S. B. Christensen, L. P. Kvist and
A. Karazmi, Planta Med., 2000, 66, 396–398.
V. S. Govindarajan, CRC Crit. Rev. Food Sci. Nutr., 1980, 12,
199–301.
N. Janaki and J. L. Bose, J. Indian Chem. Soc., 1967, 44, 985–
986.
A. Janben and T. H. Gole, J. Indian Chem. Soc., 1984, 44, 985–
986.
Y. N. Krishnamurthy, A. G. Mathew, E. S. Nambudiri,
S. Shivashankar, Y. S. Lewis and C. P. Natarajan, Trop. Sci.,
1976, 18, 37–45.
L. Peret-Almeida, A. P. F. Cherubino, R. J. Alves, L. Dufosse
and M. B. A. Gloria, Food Res. Int., 2005, 38, 1039–1044.
A. P. Gupta, M. M. Gupta and S. Kumar, J. Liq. Chromatogr.
Relat. Technol., 1999, 22, 1561–1569.
X. G. He, L. Lin, Z. L. Z. Lian and M. Lindernmaier,
J. Chromatogr., A, 1998, 818, 127.
S. J. Taylor and I. J. Mc Dowell, Chromatographia, 1992, 34,
73–77.
M. M. Naidu, B. N. Shyamala, J. R. Manjunatha,
G. Sulochanamma and P. Srinivas, J. Food Sci., 2009, 74,
C312–C318.
A. Khuranaa and C. T. Ho, J. Liq. Chromatogr. Relat. Technol.,
1988, 11, 2295–2304.
J. Zhang, S. Jinnai, R. Ikeda, M. Wada, S. Hayashida and
K. Nakashima, Anal. Sci., 2009, 25, 385–388.
D. D. Heath, M. A. Pruitt, D. E. Brenner and C. L. Rock,
J. Chromatogr. B, 2003, 783, 287–295.
M. Gonzalez, M. Gallego and M. Valcarcel, J. Agric. Food
Chem., 2003, 51, 2121–2129.
H. Yu and Q. Huang, J. Agric. Food Chem., 2011, 59, 9120–
9126.
P. Sethi, V. K. Dua, S. Mohanty, S. K. Mishra, R. Jain and
G. Edwards, J. Liq. Chromatogr. Relat. Technol., 2009, 32,
2961–2974.
A. A. D'Souza and P. V. Devarajan, J. Liq. Chromatogr. Relat.
Technol., 2013, 36(13), 1788–1801.
G. K. Jayaprakasha, G. A. N. Gowda, S. Marquez and
B. S. Patil, J. Chromatogr. B, 2013, 937, 25–32.
J. Cheng, K. Weijun, L. Yun, W. Jiabo, W. Haitao,
L. Qingmiao and X. Xiaohe, J. Pharm. Biomed. Anal., 2010,
53, 43–49.
S. Pandit, H. J. Kim, J. E. Kim and J. G. Jeon, Food Chem.,
2011, 126, 1565–1570.
W. Wichitnithad, N. Jongaroonngamsang, S. Pummangura
and P. Rojsitthisak, Phytochem. Anal., 2009, 20, 314–319.
B. K. Jadhav, K. R. Mahadik and A. R. Paradkar,
Chromatographia, 2007, 65, 483–488.
K. Inoue, C. Nomura, S. Ito, A. Nagatsu, T. Hino and H. Oka,
J. Agric. Food Chem., 2008, 56, 9328–9336.
Anal. Methods, 2014, 6, 2526–2536 | 2535

Analytical Methods
36 S. K. Sandur, M. K. Pandey, B. Sung, K. S. Ahn, A. Murakami,
G. Sethi, P. Limtrakul, V. Badmaev and B. B. Aggarwal,
Carcinogenesis, 2007, 28, 1765–1773.
37 J. Li, Y. Jiang, J. Wen, G. Fan, Y. Wu and C. Zhang, Biomed.
Chromatogr., 2009, 23, 1201–1207.
38 R. D. Jangle and B. N. Thorat, Indian J. Pharm. Sci., 2013, 75,
60–66.
39 R. Malasoni, A. Srivastava, R. R. Pandey, P. K. Srivastava and
A. K. Dwivedi, J. Sci. Ind. Res., 2013, 72, 88–97.
40 H. S. Koop, R. A. de Freitas, L. M. de Souza, G. R. Martinez
Published on 24 January 2014. Downloaded by Harvard University on 18/06/2014 22:08:30.
and J. L. M. Silveira, Chromatographia, 2013, 76, 1041–1048.
41 J. L. Quiles, M. Dolores Mesa, C. L. Ramı́rez-Tortosa,
C. M. Aguilera, M. Battino, Á. Gil and M. C. Ramı́rez-Tortosa,
Arterioscler., Thromb., Vasc. Biol., 2002, 22, 1225–1231.
42 A. M. Anderson, M. S. Mitchell and R. S. Mohan, J. Chem.
Educ., 2000, 77, 359–360.
43 R. S. Rarnsewak, D. L. DeWitt and M. G. Nair, Phytomedicine,
2000, 7, 303–308.
44 S. J. Kulkarni, K. N. Maske, M. P. Budreand and R. P. Mahajan,
Int. J. Pharmacol. Pharmaceut. Tech., 2012, 1, 81–84.
45 M. K. Kim, G. J. Choi and H. S. Lee, J. Agric. Food Chem., 2003,
51, 1578–1581.
46 The United State Pharmacopeia, United States Pharmacopeial
Convention, Rockville, MD, 24th edn, 2000.
47 M. Yang, S. Fazio, D. Munch and P. Drumm,
J. Chromatogr.,A, 2005, 1097, 124–129.
2536 | Anal. Methods, 2014, 6, 2526–2536
View Article Online
Paper
48 C. A. Hunter and K. M. Sanders, J. Am. Chem. Soc., 1990, 112,
5525–5534.
49 P. G. Stevenson, F. Gritti, G. Guiochon, K. J. Mayeld,
G. R. Dennis and R. A. Shalliker, J. Chromatogr., A, 2010,
1217, 5365–5376.
50 D. H. Marchand, K. Croes, J. W. Dolan, L. R. Snyder,
R. A. Henry, K. M. R. Kallury, S. Waite and P. W. Carr,
J. Chromatogr., A, 2005, 1062, 65–78.
51 S. Oi, Y. Ochiai and S. Miyano, Chem. Lett., 1991, 9, 1575–
1576.
52 W. H. Pirkle and Y. Liu, J. Chromatogr., A, 1996, 749, 19–24.
53 M. H. Hyun, M. S. Na and J. S. Jin, J. Chromatogr., A, 1996,
752, 77–84.
54 J. Horak, N. M. Maier and W. Lindner, J. Chromatogr., A,
2004, 1045, 43–58.
55 J. L. E. Reubsaet and R. Vieskar, J. Chromatogr., A, 1999, 841,
147–154.
56 S. Kayillo, G. R. Dennis and R. A. Shalliker, J. Chromatogr., A,
2007, 1148, 168–176.
57 M. R. Euerby, P. Petersson, W. Campbell and W. Roe,
J. Chromatogr., A, 2007, 1154, 138–151.
58 Statistics for Analytical Chemistry, 1984.
59 Separation of Curcuminoids from Turmeric – Comparison of
Polar Embedded and C18 Solid HPLC Core Columns, http://
www.separatedbyexperience.com/documents/AN-LC-Accucore-
Curcuminoids-Turmeric-AN20853_E.pdf.
This journal is © The Royal Society of Chemistry 2014