Bioscience, Biotechnology, and Biochemistry

ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20

Separation and Determination of Yellow and

Red Safflower Pigments in Food by Capillary
Electrophoresis

Toshiro Watanabe, Naoki Hasegawa, Akira Yamamoto, Shiro Nagai & Shigeru
Terabe

To cite this article: Toshiro Watanabe, Naoki Hasegawa, Akira Yamamoto, Shiro Nagai & Shigeru
Terabe (1997) Separation and Determination of Yellow and Red Safflower Pigments in Food by
Capillary Electrophoresis, Bioscience, Biotechnology, and Biochemistry, 61:7, 1179-1183, DOI:
10.1271/bbb.61.1179

To link to this article: http://dx.doi.org/10.1271/bbb.61.1179

Published online: 12 Jun 2014.

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 Biosci. Biotech. Biochem., 61 (7), 1179~ 1183, 1997

Separation and Determination of Yellow and Red Safflower Pigments in Food by

Capillary Electrophoresis
Toshiro WATANABE, Naoki HASEGAWA, Akira YAMAMOTO, Shiro NAGAI, and Shigeru TERABE*
Yaegaki Technology Development Laboratories, Yaegaki Bio-industry Inc., 681 Hayashida, Himeji, Hyogo 679-42, Japan
*Faculty of Science,
Himeji Institute of Technology, Kamigori, Hyogo 678-12, Japan
Received February 10, 1997

Capillary electrophoretic methods were developed for analyzing yellow and red safflower pigments in
food. Yellow safflower pigment was successfully separated by capillary zone electrophoresis (CZE) with a
300 mM borate buffer (pH 9.0), but this method could not successfully separate the red safflower pigment.
The red safflower pigment was discolored in an alkaline solution. Both the yellow and red pigments could
be successfully separated by micellar electrokinetic chromatography (MEK C) with 2.0% butyl acrylate/butyl
methacrylate/methacrylic acid copolymer sodium salts (BBMA), but neither pigment could be separated
with 20 mM sodium dodecyl sulfate. The yellow safflower pigment was extracted from food samples (juice
and candies) by solid-phase extraction cartridges and analyzed by the developed technique.
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Key words: capillary zone electrophoresis; micellar electrokinetic chromatography; safflower pigments

Safflower (Carthamus tinctorius L.) is indigenous to Egypt
and is known as a herb. 1) Its flowers produce red and yellow
pigments. Red safflower pigment was originally used as a
cosmetic and textile dye, and today is also used as a food
colorant. 2,3) The main component of the red pigment is
called carthamin 4 ~ 8) (Fig. 1) and, due to its low solubility
in water, the red pigment is mainly used in colored chocolate.
The yellow safflower pigment, on the other hand, has been
used as a natural food colorant for a long time,9) mainly
in colored juice, jello, and candy because of their water
solubility. The yellow pigment has numerous compo-
nents, 9 13) saffiomin A and safflor yellow B being the
main ones (Fig. 1). In recent years, natural pigments have
drawn renewed attention from the viewpoint of safety,
and an analysis method for natural pigments is thus im-
portant. However, analyzing pigments extracted from food
is very difficult because of their extremely low concentra-
tions.
Capillary electrophoresis (CE) is a relatively new
separation technique that was introduced in late 1980 and
has high separation efficiency, only requiring a small
amount of a sample. Capillary zone electrophoresis (CZE)
is the most popular separation mode for CE, where analytes
are separated on the basis of their difference in electro-
phoretic mobility, which depends on the charge and
molecular size. Micellar electrokinetic chromatography
(MEKC), in which an ionic micellar solution is employed
as the running solution, is another popular separation
mode for CE when small molecules are involve. With
MEKC, neutral analytes can be separated by their different
partitioning to the micelle. Although there are a few re-
ports on the analysis of natural pigments by high-per-
formance liquid chromatography (HPLC),2) to the best of
our knowledge, there is no analytical report on using CEo
This paper describes the extraction of safflower pigments
from food samples and their analysis by CEo
Materials and Methods
Materials. Yellow safflower pigment used commercially as a natural
coloring for food was obtained from Yaegaki Bio-industry (Himeji, Japan).
Pure water was prepared by purifying distilled water with a Milli-Q SP
system (Millipore, Bedford, MA, U.S.A.) just before use. Sodium dodecyl
sulfate (SDS) from Naca1ai Tesque (Kyoto, Japan) and butyl acrylate/butyl
methacrylate/methacrylic acid copolymer sodium salts (BBMA) from
Daiichi Kogyo Seiyaku (Kyoto, Japan) were used as anionic micellar
pseudo~stationary phases for MEKC. All other chemicals and solvents
were of analytical reagent grade and supplied by Wako (Osaka, Japan).
Apparatus. CE was performed with a BioFocus 3000 CE system
(Bio-Rad, Richmond, California, U.S.A.), using a pre-packed cartridge of
an uncoated fused silica capillary of 50 11.m i.d. and total length of 50 cm
(46 cm to the detector). HPLC separation and purification were performed
by a Beckman Gold HPLC system with a programmable 125 solvent
module and programmable 166 detector module (Fullerton, California,
U.S.A.), using an Inertsil ODS-2 packed column (4.6mm i.d. and 150mm
length with a 5-.um particle size; GL Science, Tokyo, Japan). Mass spectra
were measured with a JMS-DX303 mass spectrometer (JEOL, Tokyo,
Japan) equipped with a fast atom bombardment (F AB) ionization device.
Both negative and positive ions were measured. Glycerol was employed
as a matrix for F AB ionization.
HPLC separation. Gradient elution was performed with distilled water
containing 0.05% trifluoroacetic acid (TFA) at pH 2.5 (sol. A) and with
100% acetonitrile containing 0.05% TFA (sol. B). A linear~gradient
program from 0% to 50% of sol. B in 30 min was employed at a flow rate
of 1.0 ml/min. The wavelength of the detector was set at 400 nm for yellow
pigment and at 500 nm for red pigment. The eluate from the HPLC column
was collected when the relevant zones had been eluted, evaporated at 40°C,
and used as standard pigments for the CE analysis.
Extraction of the pigments from food samples. Yellow safflower pigment
was extracted by a solid-phase method from juice and candies. Carbonated
juice was de-aerated for 2 min in an ultrasonic bath, and candies were
dissolved in warm distilled water (60-80°C). A solid~phase extraction
cartridge (SPE ODS-4, 500 mg/6 ml; Whatman, New Jersey, U.S.A.) was
pre-conditioned with 10 m1 of methanol and then with 20 m1 of distilled
water prior to use. A 10-ml sample solution was applied to the cartridge,
and the cartridge was washed with 20 ml of distilled water. Yellow safflower
pigment was eluted with 5 ml of methanol. The eluate was evaporated to
dryness at 40°C, the resulting residue being dissolved in water and subjected
to the CE analysis.
Capillary electrophoresis (CE). A borate buffer (300 mM at pH 6.0, 7.0,
8.0, or 9.0) was used for capillary zone electrophoresis (CZE). A 20mM
SDS solution in a 50mM phosphate buffer (pH 7.0) and 2.0% BBMA
(butyl acrylate/butyl methacrylate/methacrylic acid copolymer sodium
saJts)14) in a 20mM ammonium formate buffer (pH 7.0) were used for
micellar electrokinetic chromatography (MEKC). 15) A potential of 15 kV

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 1180 T. WATANABE et al.

A
0.120
E
c
0 0.080
0
o:t
i
0
.0 0.040
«
0.000

0 5 10 15 20
Time (min)
OH OH B
Carthamin
0.120
E
c
0 0.080
0
o:t
i
0
.0 0.040
OH «
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0.000

HO 5 15 20 25 30
Time (min)
C
Safflomin A
0.120
E
c
0
0
0.080
o:t
i
0 0.040
..Q
«
0.000

10 15 20 25 30 35 40
Time (min)
Fig. 2. Separation of the Yellow Safflower Pigment by CZE.
H OH A) Capillary, 50jlm Ld. x 50cm; running solution, 300mM borate buffer at pH 7.0;
applied voltage, 15 kV; temperature, 20°C; detection, 400 nm. B) Running solution,
H H 30 mM borate buffer at pH 8.0. The other conditions are the same as those in A. C)
OH OH Running solution, 300 mM borate buffer at pH 9.0. The other conditions are the same
OH as those in A.

Safflor yellow B
Fig. 1. Structures of Red and Yellow Safflower Pigments (Carthamin,
Safflomin A and Safflor Yellow B).
was applied, detection was performed by measuring the absorbance at
400 nm (yellow pigment) or at 500 nm (red pigment), and the capillary
temperature was 20°C. Yellow safflower pigment was dissolved in distilled
water, red pigment was dissolved in N,N-dimethylformamide, each solution
then being passed through a disposable hydrophilic membrane filter
(0.45,um pore size; Advantec Toyo, Tokyo, Japan) prior to use. Each
sample was injected by pressure at 350 mbar for 1.0 s. The capillary was
rinsed with a 1.0 M NaOH solution for 120 s and with distilled water for
120s before each run.
Results and Discussion
CZE analysis
Since the yellow pigment of safflower has several diol
groups as shown in Fig. I, CZE was performed with a
borate buffer at various alkaline pH values. Good separa-
tion was obtained with a 300 mM borate buffer at pH 9.0
(Fig. 2C), although it needed a long time to complete the
separation. At pH 7.0 and pH 8.0, several components of
the yellow pigment were not well separated (Figs. 2A and
2B). The highest peak was probably a major component of
the yellow pigment of safflower. CZE separation of the red
pigment of safflower was also attempted, but the red color
faded in an alkaline solution, because the red pigment is
only stable in acidic conditions.
MEKC analysis
MEKC with a SDS solution in a phosphate buffer was
not successful for separating the yellow pigment of safflower.
A 2.0% BBMA solution in 20 mM ammonium formate (pH
7.0) gave better resolution of the yellow pigment as shown
in Fig. 3. Although the migration times are shorter in Fig.
3, the electropherogram is similar to that obtained by CZE
given in Fig. 2C. The highest peaks were probably the same
component between Figs. 2 and 3. The red pigment of
safflower was also successfully separated by MEKC with
the BBMA solution (Fig. 4). The main peak is presumed
to have been carthamin, the principal ingredient of the red
pigment of safflower. The red color of the solution did not
fade under this condition.

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 Capillary Electrophoresis of Safflower Pigments in Food 1181

0.040
RUN 5 RUN 15

0.030 0.030,.-----------, 0.030,--------,

E
c:
0
0
oo::t 0.020
i
tI)
.c
<C 0.010 0.020 0.020

E E
0.000 c c
CI CI
CI CI
In In
0 3 6 9 12 15 'lU 'lU
I/) I/)
.CI 0.010 .CI 0.010
Time (min) <C <C

Fig. 3. Separation of the Yellow Safflower Pigment by MEKC.

Running solution, 20mM ammonium formate buffer at pH 7.0 containing 2.0%
BBMA. The other conditions are the same as those in Fig. 2A.
o.ooo .........__...w._... l
0.000 .........- - -
0.030
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5 7 9 11 13 15 5 7 9 11 13 15
E 0.020 Time (min) Time (min)
c:
0 Fig. 5. Repeated Separation of Red Safflower Pigment by MEKC with
0
II)
BBMA (5th and 15th Runs).
i
II) 0.010 The conditions are the same as those in Fig. 4.
.c
<C

L~
0.040 , - - - - - - - - - - - - - - - - - - - - ,
I\.
0.000

5 7 9 11 13 15 E
c:
0 0.020
Time (min) 0
"It
Fig. 4. Separation of the Red Safflower Pigment by MEKC with BBMA. i
II)
Running solution, 20 mM ammonium formate buffer at pH 7.0 containing 2.0% .Q
BBMA; detection, 500 nm. The other conditions are the same as those in Fig. 2A. <C

Repeatability
In order to check the reproducibility for separating the
pigments by these methods, the red and yellow pigments of
safflower were repeatedly analyzed fifteen times. The yellow
pigment of safflower was analyzed by the CZE method with
a borate buffer, and the red pigment by MEKC with the
BBMA solution as already described. No significant
difference in separation pattern and migration time was
observed among the repeated runs as shown in Fig. 5 for
the red pigment.
Standard curve
The principal ingredients of the yellow pigment of
safflower isolated by HPLC were employed as standards
for the yellow pigment by CEo The yellow pigment of
safflower prepared by HPLC gave a single major peak (Fig.
6). The main component in Fig. 6 showed the same
migration time as the highest peak in Fig. 2C, and it is
hence concluded to be the principal component of the yellow
pigment of safflower. A standard curve obtained with this
component from 50 to 1000 ppm showed a straight line
(r = 0.998), the detection limit for the yellow pigment being
0.5 ppm. The yellow pigment component of safflower was
determined with this standard curve.
Mass spectrometric analysis
Mass spectra for the principal component of the yellow
0.000 1 1 .,.,.,- "
10 15 20 25 30 35 40
Time (min)
Fig. 6. Separation of the Principal Ingredient of Yellow Safflower
Pigment by CZE.
The conditions are the same as those in Fig. 2C.
safflower pigment isolated by HPLC were measured in both
the positive and negative ion modes by F ABMS. The
molecular mass of the principal component was determined
to be 612 from the m/z 613 peak for M + 1 in the positive
ion mode and m/z 611 peak for M 1 in the negative ion
mode (Fig. 7). The molecular mass strongly suggests that
the principal component of the yellow pigments was
safflomin A as suggested in earlier reports. 9 ,16)
Analysis of the pigments extracted from food samples
Pigments were extracted from candies as described in
Materials and Methods, and juice with a dark color was
analyzed without any pretreatment. The recovery of the
pigments by solid-phase extraction was 75%. An elec-
tropherogram of the pigments extracted from candies (Fig.
8B) was similar to that of the yellow pigment (Fig. 2C). In
the electropherogram (Fig. 8A) of the pigment extracted
from juice, many peaks appeared, among which the peak
at about 21 min was easily identified as safflomin A.

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 1182 T. WATANABE et al.

A A
183~5-'

1 0.040

E
c
256 c
C
"It
611 0.020
I 'S
III
..Q
C(
551 591 703
0.000
100 200 300 400 500 600 700
m/e
10 15 20 25 30 35 40
Time (min)
B
B
553
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500 ji ~1361r 737

0.120

Gi 2~3: 258 369

IIt.m,'it'b"t,yj';
X 10 I
~ 0.080
a: 20 . 294~28 t
461 8"It
407442
O~. .~~. ..w~~~~.-----~~--~~ 'S
100 200 300 400 500 600 700 ! 0.040
m/e C(

Fig. 7. FABMS Spectrum of the Principal Ingredient of Yellow Safflower

o.ooo~--
Pigment.
A, negative ions; B, positive ions.
10 15 20 25 30 35 40
Time (min)

Fig. 8. CZE Separation of Pigments Extracted from Food Samples.

A, yellow safflower pigment extracted from juice; B, yellow safflower pigment
extracted from candies. The conditions are the same as those in Fig. 2C.

Table Analytical Results for the Yellow Pigment Extracted from Food Samples

Migration time Determination

n
Species
(Intra-day)
Mean (min) C.V. (%) C.V. (%)

Standard
Safflower yellow* 21.62 0.16 1380 1.87
Yellow pigment extracted from food samples
From juice* 3 21.33 0.26 427 2.36
From candies* 3 21.17 0.45 1253 0.94

* Safflomin A.

However in Fig. 8A, a number of extra peaks are also of the CE method is its low running cost. The CE method
apparent other than those in the yellow safflower pigment will also be widely applicable to analyzing several different
(Fig. 2C), suggesting that some pigments other than that food additives.
of safflower pigments had also been added to the juice. The
coefficient of variation (C.V.) of the migration times was References
less than 0.5% and that for quantification was less than 1) K. Saito, Lebensm. Unters. Forsch., 192, 343-347 (1991).
2.5% (Table). 2) K. Nakano, Y. Sekino, N. Yomo, S. Wakayama, S. Miyano, K.
In conclusion, CE was found to be a useful technique Kusaka, and E. Daimon, J. Chromatogr., 438, 61-72 (1988).
for analyzing food pigments. The amount of the sample 3) K. Saito and T. Murata, Lebensm. Unters. Forsch., 198, 277-283
(1994).
required for the analysis was very small, and the repeat-
4) K. Saito and K. Miyakawa, Lebensm.-Wiss. u.-Technol., 27, 384--385
ability of migration times and quantification was high (1994).
even for real samples, probably due to the absence of any 5) K. Saito, Y. Takahashi, and M. Wada, Biochim. Biophys. Acta, 756,
packing material inside the capillary. Another advantage 217-222 (1983).

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 Capillary Electrophoresis of Safflower Pigments in Food
6) K. Saito and A. Fukushima, Food Chern., 29,161-175 (1988).
7) K. Saito, T. Murata, and T. Mori, Int. l. Food Sci. Technol., 29,
715-719 (1995).
8) K. Saito and T. Mori, Food Res. Int., 27. 571-574 (1994).
9) J. Onodera, H. Obara, M. Osone, Y. Maruyama, and S. Sato, Chern.
Lett., 3, 433--436 (1981).
10) J. Onodera, H. Obara, R. Hirose, S. Matsuba, N. Sato, S. Sato, and
M. Suzuki, Chern. Lett., 9,1571-1574 (1989).
11) K. Saito and T. Murata, Food Chern., 5t, 307-310 (1994).
12) S. Sato, H. Obara, 1. Onodera, A. Endo, and S. Matsuba, Bull. Chern.
Downloaded by [178.138.34.96] at 11:36 16 November 2017
1183
Soc. lpn., 65, 452-457 (1992).
13) Y. Takahashi, N. Miyasaka, S. Tasaka, I. Miura, S. Vrano, M. Ikura,
K. Hikichi, K. Matsumoto, and M. Wada, Tetrahedron Lett., 23,
5163-5166 (1982).
14) H. Ozaki, S, Terabe, and A. Ichihara, J. Chrornatogr. A, 680, 117-123
(1994).
15) S. Terabe, Trends Anal. Chern., 8, 129-134 (1989).
16) 1. Onodera, K. Kawamoto, S. Matsuba, S. Sato, H. Kojima, Y.
Kaneya, and H. Obara, Chern. Lett., 1995, 901-902.
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