Food Chemistry 174 (2015) 380–386

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Aqueous two-phase based on ionic liquid liquid–liquid microextraction

for simultaneous determination of five synthetic food colourants
in different food samples by high-performance liquid
chromatography
Ou Sha a,b,c, Xiashi Zhu a,⇑, Yanli Feng c, Weixing Ma c
a
College of Chemistry & Chemical Engineering, Yangzhou University, Yangzhou 225002, China
b
Analysis and Test Centre of Jiangsu Marine Resources Development Research Institute, Lianyungang 222001, China
c
School of Chemistry and Chemical Engineering, Huaihai Institute of Technology, Lianyungang 222005, China

a r t i c l e i n f o a b s t r a c t

Article history: A rapid and effective method of aqueous two-phase systems based on ionic liquid microextraction for the
Received 20 April 2014 simultaneous determination of five synthetic food colourants (tartrazine, sunset yellow, amaranth, pon-
Received in revised form 6 August 2014 ceau 4R and brilliant blue) in food samples was established. High-performance liquid chromatography
Accepted 11 November 2014
coupled with an ultraviolet detector of variable wavelength was used for the determinations.
Available online 15 November 2014
1-alkyl-3-methylimidazolium bromide was selected as the extraction reagent. The extraction efficiency
of the five colourants in the proposed system is influenced by the types of salts, concentrations of salt
Keywords:
and [CnMIM]Br, as well as the extracting time. Under the optimal conditions, the extraction efficiencies
Synthetic food colourants
Ionic liquid
for these five colourants were above 95%. The phase behaviours of aqueous two-phase system and extrac-
HPLC tion mechanism were investigated by UV–vis spectroscopy. This method was applied to the analysis of
Aqueous two-phase the five colourants in real food samples with the detection limit of 0.051–0.074 ng/mL. Good spiked
recoveries from 93.2% to 98.9% were obtained.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction (Oka et al., 1994), high-performance liquid chromatography (HPLC)

Synthetic or natural food colourants are usually added to
foodstuffs to make them more visually attractive to consumers
and to restore their original appearance when it has been lost dur-
ing production processes (Kucharska & Grabka, 2010). Studies
demonstrate that the chronic consumption of synthetic colourants
seems to predispose to neurotoxicity in young and adult animals
and some synthetic food colors could be reduced by azoreductase
enzymes in intestinal bacteria and in liver cells with the release
of aromatic amines to the organism (Hildenbrand, Schmahl,
Wodarz, Kimmel, & Dartsch, 1999; Nagaraja & Desiraju, 1993).
Considering both the potential effects on human health and the
need for knowledge of the components of food, detection of syn-
thetic dyes in different food samples is an important issue to deal
with. Various analytical techniques, included spectrophotometric
methods (Turak & Ozgur, 2013), voltametric methods (Mo et al.,
2010), differential pulse polarography (Chanlon, Joly-Pottuz,
Chatelut, Vittori, & Cretier, 2005), thin layer chromatography
⇑ Corresponding author. Tel./fax: +86 518 85856483.
E-mail address: 7993259@163.com (X. Zhu).
and so on (Minioti, Sakellariou, & Thomaidis, 2007; Pereira Alves,
Brum, Branco de Andrade, & Pereira Netto, 2008), have been
developed to facilitate the simultaneous determination of various
synthetic food colourants. Preconcentration method was usually
used combined with these techniques to minimise potential
interferences from diversified components present in food samples
and concentrate the analyte from a low content of food sample
(El-Shahawi, Hamza, Al-Sibaai, Bashammakh, & Al-Saidi, 2013;
Pourreza & Ghomi, 2011; Shen, Zhang, Prinyawiwatkul, & Xu,
2014).
Aqueous two-phase system (ATPS) is a very promising technol-
ogy for separation and pretreatment (Tang et al., 2014; Jiang, Lu,
Tan, Liang, & Cui, 2014). Compared with the traditional organic sol-
vent extraction and solid phase extraction, ATPS is considered to be
environmentally friendly due to the bulk of both phases consist of
water and no use of volatile organic solvent in the whole process.
Since a new type of ATPS consisting of ionic liquid (IL) and salts
were reported in 2003 by Rogers and co-workers for the first time,
the IL–salt ATPS (IL–ATPS) has been applied to the separation and
preconcentration of organic compounds, inorganic compound and
biomolecules in different matrices (He, Li, Liu, Li, & Liu, 2005;

http://dx.doi.org/10.1016/j.foodchem.2014.11.068
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
 O. Sha et al. / Food Chemistry 174 (2015) 380–386
Passos et al., 2013; Tan, Li, & Xu, 2012; Yang et al., 2014). The
IL–salt ATPS (IL–ATPS) has many advantages, such as low viscosity,
little emulsion formation, no need of using volatile organic solvent,
quick phase separation, high extraction efficiency, and gentle bio-
compatible environment.
In this paper, an IL–ATPS based on hydrophilic ionic liquid,
1-alkyl-3-methylimidazolium bromide ([CnMIM][Br]), and salt
was established to extract and determine the commonest five
synthetic food colourants, tartrazine (Ta), sunset yellow (SY), ama-
ranth (Am), ponceau 4R (Pon) and brilliant blue (BB), in different
food samples with HPLC. The phase equilibrium of IL–ATPS and
the factors on the extraction of colourants, such as the types of
salts and IL, concentrations of salt and [CnMIM][Br], as well as
the extracting time and centrifugation time were discussed in
detail. The [C4MIM]Br-salt ATPS was not easy to emulsify and
[C4MIM]Br was diffluent in water and methanol (HPLC mobile
phase). Colourants could be extracted into IL phase and analysed
by HPLC with direct sampling after ATPS extraction. Under the
optimum conditions, the proposed method was applied to precon-
centration colourants in real food samples. In addition, the
mechanism of IL–ATPS formation was discussed, and the extrac-
tion mechanism of the IL–ATPS was investigated by UV–vis
spectroscopy.
2. Experimental
2.1. Apparatus
An Agilent 1260 HPLC system (Agilent, USA), equipped with a
1260 infinity quaternary pump, and a 1260 infinity variable
wavelength detector (190–600 nm), was used for colourants
determination. Chromatographic separation was achieved on a
Eclipse plus-C18 column (4.6 mm  150 mm  3.5 lm) (Agilent,
USA). All spectra measurements were carried out by using a model
UV-2501 spectrophotometer (Shimadzu, Japan); all pH values were
measured by a PHS-25B pH-metre (Shanghai Precision & Scientific
Instrument Co., Ltd., Shanghai, China) and the phase separation
was assisted with a TG16-WS centrifuge (Hunan Xiangyi Centifuge
Instrument Co., Ltd., Chang sha, China).
2.2. Standard solutions and reagents
The standard stock solutions of the colourants, tartrazine (Ta;
C.I. Food yellow 4; 0.5 mg/mL), amaranth (Am; C.I. Food red 9;
0.5 mg/mL), sunset yellow (SY; C.I. Food yellow 3; 0.5 mg/mL),
ponceau 4R (E124) (Pon; C.I. Food red 7; 0.5 mg/mL), brilliant blue
(BB; C.I. Food blue 2; 0.5 mg/mL) were obtained from the National
Research Center for Certified Reference Materials (Beijing, Chi-
na).The mixed standard solutions containing all colourants at
20.0 lg/mL was prepared by mixing and dilution of appropriate
aliquots from standard stock solution of each substance. Working
solutions were prepared by appropriate dilutions of the mixed
standard solutions with water.
HPLC-grade methanol was purchased from Merck KGaA Co., Ltd.
(Germany). 1-butyl-3-methylimidazolium bromide ([C4MIM][Br]),
1-hexyl-3-methylimidazolium bromide ([C6MIM][Br]), 1-octyl-
3-methylimidazolium bromide ([C8MIM][Br]) and 1-decyl-3-meth-
ylimidazolium bromide ([C10MIM][Br]) were obtained from
Shanghai Cheng Jie Chemical Co., Ltd. (Shanghai, China). 0.3 g/mL
[CnMIM]Br aqueous solution was prepared by distilled water.
K2HPO43H2O was used as the salt for the aqueous two-phase
systems. Milli-Q water (Millipore, Bedford, MA, USA) was used
throughout the study. Britton–Robinson (B–R) buffer solutions
were prepared by mixing the mixed acid (composed of
0.04 mol L1 H3PO4, HAc and H3BO3) with 0.2 mol L1 NaOH in
381
proportion. Unless otherwise specified, all other reagents were
analytical grade and were purchased from Sinopharm Chemical
Reagent Co., Ltd. (Shanghai, China).
2.3. Preparation of phase diagram
Phase diagrams were determined by the means of cloud-point
method (Li, He, Liu, Li, & Liu, 2005). A certain of [CnMIM]Br was
put into a 10.0 mL centrifugal tube. The tested salt solution was
added dropwise to the test tube until turbidity. Whereafter, a
two-phase system was achieved and water was added dropwise
to the test tube to obtain a clear one-phase system. More salt
solution was added again to afford a two-phase system. The
composition of this mixture was noted and so on. The bimodal
curve was applied to characterise the phase diagram (Vollhardt &
Fainerman, 2010). In the region above the bimodal curve, the
system is divided into two phases; in the region below the bimodal
curve, the system is of a homogeneous phase.
2.4. Preparation of the sample solution
All samples, including soft drink, sugar-based, instant powdered
drink and gelatin-based confectionery, were obtained from a local
market. Appropriate amounts (0.1–5 g) of the samples were
dissolved in 15 mL of water. The carbonated drinks were degassed
by ultrasonication for 5 min to remove the carbon dioxide. A
warming process (50 °C, 30 min) was used for the complete
dissolution of the sugar-based, instant powdered drink and
gelatin-based confectionery. These solutions were centrifuged
and the upper solutions were filtered through 0.45 lm micro-pore
filter membrane. The filtrate was transferred to volumetric flask of
25.0 mL and completed to the mark with distilled water and
prepared for IL–ATPS extraction.
2.5. Extraction procedure
0.5 mL of 0.3 g/mL [C4MIM]Br, 0.50 mL B–R buffer solution and
a certain of the mixed standard solution or the sample solution
were placed in a 10.0 mL centrifugal tube. The mixture was diluted
to 5.0 mL with distilled water and then 5.5 g K2HPO43H2O was
added. A turbid solution was easily formed after gentle blending.
The tube centrifuged for 4 min at 3000 rpm to ensure a complete
phase separation. The upper aqueous phase was removed with a
syringe, and the volume of the IL phase collected was almost
120 lL. Then 20.0 lL of [C4MIM]Br phase was injected into HPLC
for quantification.
2.6. Phase behaviour of IL-ATPS
The extraction efficiency depends on the structure of analyte
and its affinity towards the extractant (described as partitioning
coefficient), phase ratio and the number of extraction in the
liquid–liquid extraction system. Among these factors, partitioning
coefficient K is the most important physical and chemical parame-
ter for the study of phase behaviour. Higher separation efficiency
can be achieved when a greater partitioning coefficient K and a
lower phase ratio R were selected (Wuhan University, 1995). In
this paper, quantitative extraction of the five colourants was
accomplished with a one-step extraction by using IL–ATPS. So
the distribution behaviours of these colourants between IL phase
and salt phase were characterised by the extraction efficiency (E),
partition coefficient (K) and phase ratio (R).
The parameters E, K and R were defined as follows:
C IL V IL
E¼  100% ð1Þ
C aq V aq þ C IL V IL
382 O. Sha et al. / Food Chemistry 174 (2015) 380–386

Pon at 254nm adding appropriate amount of different salts, such as (NH4)2SO4,

Absorbance/mAu

Ta Na2SO4, K2HPO4. But, those salts, such as K3PO4, KH2PO4, NaCl,

Am SY NaH2PO4, cannot separate [C4MIM]Br solution into two phases. It
was found that the phase separation ability of salts followed:
K2HPO4 > (NH4)2SO4 > Na2SO4. This order was consistent with their
BB solubility in water at 25 °C, suggesting that the difference in phase
separation capability was due to distinct solubility in water
(Deanm, 1985). Therefore K2HPO4 was chosen in the following
t/minute studies.
Ta at 420nm
SY
Pon Am at 510nm
Absorbance/mAu

Ta
Pon at 520nm 3.2. The selection of ionic liquid
Am SY at 480nm
bb at 590nm
BB Methylimidazolium bromides with four different carbon-chain
length, namely [C4MIM]Br, [C6MIM]Br, [C8MIM]Br and [C10MIM]Br,
were selected to investigate the effect of carbon-chain length on
phase diagrams of IL–ATPS. The bimodal curves determined at
t/minute
25 °C for the IL/K2HPO4 system were investigated. When the
weight percentage of K2HPO4 was more than 0.15%, the phase
Fig. 1. HPLC chromatograms of mixed food colourant standard solutions. The separation capability of IL followed the order of [C4MIM]Br 
concentration of the each colourant was 4.0 lg/mL. [C6MIM]Br  [C8MIM]Br < [C10MIM]Br. In view of the lower
synthetic cost and lower viscosity of [C4MIM]Br than that of
[C10MIM]Br, [C4MIM]Br was chosen for further study.
C IL
K¼ ð2Þ
C aq
3.3. Effect of sample pH

V IL The effect of pH on IL–ATPS extraction ability of [C4MIM]Br for

R¼
V aq
where, CIL and Caq are the concentration of the five colourants in IL
phase and in salt-aqueous phase; VIL and Vaq are the volume of IL
phase and salt-aqueous phase respectively.
The peak area of these five colourants in [C4MIM]Br/K2HPO4
system were determined as an example to study the distribution
behaviour and extraction efficiency. After the IL–ATPS extraction
the IL phase was collected and diluted to 1.0 mL with distilled
water to measure the peak area of these five colourants by the
Agilent 1260 HPLC system. The solution containing the same con-
centration of colourants without extraction was used as reference
solution. Therefore E, K and R could be calculated with Eqs. (1)–(3).
2.7. Chromatographic conditions
The mobile phase A contained 0.02 mol/L ammonium acetate
aqueous solution (pH 4.5, adjusted by glacial acetic acid). Mobile
phase B was methanol. The gradient conditions were 15–50% B
(0–6 min), followed by 50–35% B (6–10 min), and changed to
35–15% B at last (10–15 min). A flow rate of 0.8 mL/min was used
throughout the 15-min run. The temperature of the column was
controlled at 30 °C. Ultraviolet detection of variable wavelength
was used. In order to reduce the interference affected by other
organic substance in the food sample at 254 nm and got a higher
detectable signal for brilliant blue, the detection wavelength of
HPLC was set at the maximum absorption wavelength of 420 nm
for tartrazine, 480 nm for sunset yellow, 510 nm for amaranth,
520 nm for ponceau 4R and 590 nm for brilliant blue respectively
(Fig. 1). The mobile phase was filtered through 0.45 lm micro-pore
filter membrane prior to use.
3. Results and discussion
3.1. The selection of salt
The bimodal curve for the aqueous two phase systems of
[C4MIM]Br with a series of salts at 25 °C was investigated for the
selection of salt. Two replicate measurements were performed
for each point. Results showed that IL–ATPS could be formed by
ð3Þ
these five colourants was studied in the pH range of 3.0–10.0 by
the addition of Britton–Robinson buffer solution. It was found that
peak area of all colourants remained relatively constant over the
pH range of 3.0–10.0. In this work, pH 6.0 was chosen for further
work considering that most food and cosmetic samples are weak
acids or neutral.
3.4. Effect of the amount of [C4MIM]Br
The amount of [C4MIM]Br used in the preconcentration proce-
dure is a critical factor for obtaining a high extraction performance.
Therefore, the extraction system was carefully studied to deter-
mine the lowest IL-phase volume necessary for achieving the best
extraction. The effect of 0.3 g/mL [C4MIM]Br aqueous solution on
the peak area, phase ratio and the partition coefficient was studied
within the range of 0.10–0.70 mL. It was observed that the peak
area increased in the concentration range of 0.10–0.50 mL and
remained constant above that (Fig. 2a). The phase ratio increased
with the increased amount of [C4MIM]Br due to the more
[C4MIM]Br was driven into the upper phase where the enriched
[C4MIM]Br existed and the partition coefficient decreased when
the amount of [C4MIM]Br was above 0.5 mL (Fig. 2b). The extrac-
tion efficiency calculated was above 95% in the concentration
range of 0.50–0.70 mL. Considering the extraction efficiency,
enrichment factors and the low consumption of [C4MIM]Br,
0.5 mL of 0.3 g/mL [C4MIM]Br solution was used to achieve a
higher enrichment factor in the subsequent experiments.
3.5. Effect of salt
The influence of K2HPO4 concentration on the peak area was
studied and the results obtained were shown in Fig. 4. Peak area
of the five synthetic food colourants increased with the amount
of K2HPO4 up to 5.5 g and remained unchanged (Fig. 3a). The
extraction efficiency calculated was above 95% in the concentra-
tion range of 5.50–7.00 g. It revealed in Fig. 3b that phase ratio
increased in the range of 4.5–7.0 g and partition coefficient got a
maximum value when the amount of K2HPO4 was 5.5 g. In order
to achieve quantitative extraction and higher separation efficiency,
5.5 g of K2HPO4 was used in all further experiments.
 O. Sha et al. / Food Chemistry 174 (2015) 380–386 383

(a) (b)
R

Peak area
K

R
K
Tartrazine Tartrazine
Amaranth Amaranth
Ponceau 4R Ponceau 4R
Sunset yellow Sunset yellow
Brilliant blue Brilliant blue

IL/mL IL/mL

Fig. 2. Effects of [C4MIM]Br amount on peak area, phase ratio and partition coefficient. Conditions: concentration of colourants was 0.5 lg/mL respectively, 5.5 g
K2HPO43H2O, 0.50 mL pH 6.0 B–R buffer solution.

(a) (b) R
Peak area

R
K

Tartrazine K
Ponceau 4R Tartrazine
Amaranth Amaranth
Sunset yellow Ponceau 4R
Brilliant blue Sunset yellow
Brilliant blue

Salt/g Salt/g

Fig. 3. Effects of salt amount on peak area, phase ratio and partition coefficient. Conditions: concentration of colourants was 0.5 lg/mL respectively, 0.5mL 0.3 g/mL
[C4MIM]Br; 0.50 mL pH 6.0 B–R buffer solution.

5,6,7
1—— 4.8 g K2HPO4+0.3mL[C4min]Br 3.6. Effect of extraction time and centrifugation time
2—— 5.0 g K2HPO4+0.3mL[C4min]Br
4 3—— 5.5 g K2HPO4+0.3mL[C4min]Br
4—— 6.0 g K2HPO4+0.3mL[C4min]Br The effect of extraction time was examined within the range of
3 5—— 7.5 g K2HPO4+0.3mL[C4min]Br 0–10 min at room temperature. In this experiment, extraction time
6—— 8.0 g K2HPO4+0.3mL[C4min]Br
2 7—— 0.0 g K2HPO4+0.3mL[C4min]Br was from the salt addition into the solution after manual shaking
to before the initiation of centrifugation. It was found that a turbid
solution was easily formed when certain amount of salt was added
to the system at temperature and the extraction time had little sig-
e
Absorbance

1
nificant effect on the extraction efficiency. Then the turbid cloudy
e' solution was centrifuged at 3000 rpm for 4 min to completely
f
reach a clear separation of two phases. Therefore, to keep the anal-
f' ysis time as short as possible, the cloudy solution was centrifuged
for 4 min immediately after the preparation at room temperature.

a
a'
b
b' c
3.7. Mechanism investigation
c'
d
d' It is important and necessary to know the phase formation and
the speciation of analyte in the IL–ATPS (Ruiz-Ruiz, Benavides,
Wavelength/nm Aguilar, & Rito-Palomares, 2012). One can either take advantage
of the extraction mechanisms which ILs afforded, or afford optimal
Fig. 4. Absorbance spectra of [C4MIM]Br in top phase for various concentrations of
K2HPO4 and the spectra of different colourants standard solution before and after separation condition and accurate analysis for analyte. In this
extraction process. a,a0 – tartrazine; b,b0 – sunset yellow; c,c0 – amaranth; d,d0 – paper, the phase formation mechanism and speciation analysis of
ponceau 4R; e,e0 – brilliant blue; f,f0 – the mixture solution. the five colourants in IL–ATPS were investigated.
384 O. Sha et al. / Food Chemistry 174 (2015) 380–386

3.7.1. Phase formation mechanism of IL/ATPS
Hydrophil ionic liquid and salt are the important factors in the
phase formation of IL-APTS. Hydrophil ionic liquid tend to be
water-destructuring salts and was capable of being salted-out by
water-structuring salts (Bridges, Gutowski, & Rogers, 2007). The
mechanism of IL–ATPS formation is under the label of hydration
theory (Trindade et al., 2007). In this study, the spectra of
[C4MIM]Br phase without analytes added was investigated with
UV–vis spectroscopy. With a fixed volume of 0.3 g/mL [C4MIM]Br
of 0.5 mL, the spectra of [C4MIM]Br phase with different amount
of K2HPO4 added was scanned in the range of 190.0–300.0 nm
(Fig. 4). It could be seen that the maximum absorbance of the solu-
tion of [C4MIM]Br phase increased with the increasing amount of
K2HPO4 (Curve 1 ? Curve 5). When the amount of K2HPO4
exceeded 7.5 g, the maximum absorbance was invariable and the
spectra of the solution of [C4MIM]Br phase was practically super-
imposed (Curve 5 ? Curve 6). The spectra of 0.5 mL [C4MIM]Br
solution without K2HPO4 added (Curve 7) was coincide with that
of the solution of [C4MIM]Br phase with 7.5 g K2HPO4 added (Curve
5). This means that almost all [C4MIM]Br was enriched in the top
phase with the increased amount of inorganic salt. These results
revealed the formation of IL–ATPS may be considered to be a com-
petition between the hydrophilic IL and the inorganic salt for the
water molecules. The competition is won by the inorganic ions
because of their stronger affinity for the water. In other words,
there is a ‘‘migration’’ of water molecules away from the ions of
the IL to those of the inorganic salt, which, in turn, decreases the
hydration and hence the solubility of the ions of the IL. Conse-
quently, a phase rich in the salted-out IL separates from the rest
of the solution. This means that the salting-out effect must be
directly correlated to the hydration strength of the different ions
of the inorganic salt.
3.7.2. Spectrometric studies of the colourants in the [C4MIM]Br-rich
top phase
The absorption spectra of tartrazine, sunset yellow, amaranth,
ponceau 4R, brilliant blue alone and in the mixture before and
after [C4MIM]Br/K2HPO4 ATPS extraction were scanned against
the reagent blank respectively in the wavelength range of
350–700 nm (Fig. 4).The absorption spectra recorded for tartrazine
(curve a and curve a0 ), amaranth (curve b and curve b0 ), ponceau 4R
(curve c and curve c0 ), sunset yellow (curve d and curve d0 ), brilliant
blue (curve e and curve e0 ) and the mixture solution (curve f and
curve f0 ) before and after IL/salt ATPS extraction showed that their
maximum absorption occur at 420 nm, 510 nm, 520 nm, 480 nm
and 630 nm respectively. It can be seen in Fig. 4 that the spectra
of these colourants was remained unaltered when colourants were
extracted into the [C4MIM]Br-rich top phase and practically super-
imposed before and after [C4MIM]Br/K2HPO4 ATPS extraction. This
means that no direct chemical (bonding) interactions were
involved between colourants and ionic liquid in the IL–ATPS and
the speciation of these colourants was not changed before and
after [C4MIM]Br/K2HPO4 ATPS extraction.
3.8. Analytical performance
The analytical performance of this method was evaluated under
the optimised conditions. The calibration equations and the corre-
lation coefficient for Ta, Am, Pon, SY and BB were as follows:
A = 2462.5C (lg/mL) + 38.4, R2 = 0.9996; A = 2551.2C (lg/mL) +
19.6, R2 = 0.9997; A = 1894.5C (lg/mL)+28.7, R2 = 0.9991;
A = 2739.4C (lg/mL) + 31.5, R2 = 0.9991; A = 2211.2C (lg/mL) +
19.1, R2 = 0.9992. Detection limits were calculated following
American Chemical Society guidelines (ACS, 1980), and was
found to correspond to the analyte concentrations equivalent to

Table 1
Comparison of analytical performance of the proposed method with other methods.

Preconcentration Detection technique Linearity Detection limit References

1 Cloud point extraction UV–vis 0.05–3.50 lg/mL (BB) 0.017 lg/ml (BB) Pourreza and Ghomi (2011)
2 Ultrasound-assisted solvent extraction HPLC – 10 ng/mL (Ta) Shen et al. (2014)
20 ng/mL (Am)
10 ng/ml (SY)
3 Ionic liquid dispersive phase HPLC 0.5–2000 ng/mL (Ta) 0.15 ng/mL (Ta) Wu et al. (2013)
microextraction 0.05–300 ng/mL (Am) 0.017 ng/mL
(Am)
0.05–300 ng/mL (SY) 0.015 ng/mL (SY)
0.05–300 ng/mL 0.015 ng/mL
(Pon) (Pon)
4 Without extraction Derivative 2–10 lg/mL (BB) 0.18 lg/mL (BB) Turak and Ozgur (2013)
spectrophotometry
5 Without extraction Electrochemical sensor 9–549 lg/L (Ta) 2.7 lg/L (Ta) Gan, Sun, Meng, Song, and Zhang
9–927 lg/L (SY) 3.7 lg/L (SY) (2013)
6 Aqueous two-Phase UV–vis 0.25–750 ng/mL (BB) 0.12 ng/mL (BB) Shiri, Khezeli, Lotfi, and Shiri (2013)
7 Without extraction Spectrophotometry 0–10.0 mg/L (Ta) – El-Sheikh and Al-Degs (2013)
0–9.0 mg/L (SY) –
8 Without extraction UV–vis 1–25 lg/mL (BB) – Sorouraddin, Rostami, and Saadati
2–40 lg/mL (SY) (2011)
2–40 lg/mL (Ta)
9 Aqueous two-Phase based on IL HPLC 0.61–2000 ng/mL 0.053 ng/mL (Ta) This work
microextraction (Ta)
0.81–2000 ng/mL 0.056 ng/ML
(Am) (Am)
0.83–2000 ng/mL 0.074 ng/ML (PR)
(Pon)
0.58–2000 ng/mL 0.051 ng/Ml (SY)
(SY)
0.81–2000 ng/mL 0.063 ng/Ml (BB)
(BB)
 O. Sha et al. / Food Chemistry 174 (2015) 380–386 385

Table 2
Determination of colourants in different food sample from the local market (n = 3).

Sample Colourant Concentration before Added/(ng/mL) Founded/(ng/mL) Recovery/% Content/mg/kg

extraction/(ng/mL)
Carbonated drink-green Tartrazine 240.4 200 189.6 94.8 12.02
Brilliant blue 23.6 20 19.4 97.0 0.59
Carbonated drink-orange Ponceau 4R 6.1 10 9.6 96.0 1.53
Tartrazine 33.1 30 28.4 94.7 8.27
Sunset yellow 49.0 50 47.9 95.8 12.26
Carbonated drink-purple Amaranth 185.2 200 190.3 95.2 9.26
Brilliant blue 10.8 10 9.65 96.5 0.54
Lollipop Tartrazine 49.2 50 48.1 96.2 1.23
Brilliant blue 2.8 5 4.74 94.8 0.07
Fruit-flavoured jelly Tartrazine 182.4 200 189.5 94.8 4.56
Ponceau 4R 4.4 5 4.73 94.6 0.11
Amaranth 9.2 10 9.87 98.7 0.23
Fruit-flavoured candy Ponceau 4R 6 10 9.54 95.4 0.15
Milk tea powder Tartrazine 429.0 400 376.5 94.1 21.45
Ponceau 4R 1.8 2 1.89 94.5 0.09
Papaya powder Tartrazine 152.7 200 186.4 93.2 19.09
Ponceau 4R 79.1 100 94.8 94.8 9.89
Chocolate candy-yellow Tartrazine 845.8 800 788.6 98.6 211.45
Sunset yellow 2.5 5 4.76 95.2 0.63
Chocolate candy-green Tartrazine 819.0 800 791.3 98.9 40.95
Sunset yellow 8.4 10 9.51 95.1 0.42
Brilliant blue 9.2 10 9.48 94.8 0.46

signal-to-noise ratios of three. The linearity and detection limits for
the five colourants were seen in Table 1. The relative standard
deviations (RSDs) were 2.3% (Ta), 3.2% (Am), 2.4% (Pon), 1.6% (SY)
and 2.5% (BB) (n = 5, c = 0.50 lg/mL) respectively. The enhance-
ment factors, defined as the ratio of slope of preconcentrated sam-
ples to that obtained without preconcentration, were 43 (Ta), 49
(Am), 32 (Pon), 42 (SY) and 40 (BB) respectively.
Compared with the previously reported methods for the deter-
mination of colourants in food samples, the proposed method is
effective for the determination of colourants with lower limit
detection and wider linearity than majority of the reported meth-
ods (Table 1).
3.9. Application of real samples
The developed method was used to determine the colourants in
different food products from the local market. The samples were
prepared as described in Section 2.4. In order to assess the accuracy
of the proposed method, recovery studies were also performed for
the corresponding samples by determining known amounts of
colourants added to the samples. Table 2 shows that the recoveries
of the colourants ranged from 93.2% to 98.9%.
4. Conclusion
A simple and rapid IL–ATPS consisting of [C4MIM]Br and
K2HPO4 coupled with HPLC–UV was developed for the sensitive
simultaneous determination of five synthetic food colourants in
different food samples. In this IL–ATPS, the mechanism of IL–ATPS
formation was discussed by hydration theory. In the process
of extracting, chemical interactions between colourants and
[C4MIM]Br were not observed by UV–vis spectroscopy. The
developed method proved to be highly efficient, fast, non-toxic
and simple for the separation and enrichment of colourants in food
samples. This method might provide the potential in the concen-
tration and separation of other food samples.
Acknowledgements
The authors acknowledge the financial support from the Fund
Project of National Natural Science Foundation of China
(20875082; 21155001), China; the Fund Project of Science and
Technology of Jiangsu Marine Resources Development Research
Institute (JSIMR201309), China; the Fund Project of Priority
Academic Program Development of Jiangsu Higher Education
Institutions, China.
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