tartrazine ( ta ) and sunset yellow ( sy ) are synthetic food colors used most extensively as food additives , to improve the appearance , color , and texture of foods . when added in excess , however , these synthetic food colors can be pathogenic . to ensure food safety
, the chinese government has imposed rigorous standards on the permitted levels for various synthetic food colors .
for the same reason , it is important to develop effective methods for analyzing synthetic colors in food .
various techniques have been applied for analyzing synthetic colors in food samples , including spectrophotometry , capillary electrophoresis , differential pulse polarography , liquid chromatography , and others , which all require an optimal extraction method to concentrate an analyte from a small amount of food sample containing miscellaneous components [ 9 , 10 ] .
solid phase extraction ( spe ) and liquid - liquid extraction ( lle ) have been reported for separating synthetic food colors from different matrices [ 11 , 12 ] .
for spe , analytes are separated following complicated and time - consuming absorption and desorption steps involving the use of toxic and volatile reagents , such as methanol , acetic acid , and ammonia , that are used in the current chinese national standard method . in contrast , an aqueous two - phase system ( atps ) has attracted increasing attention in that it minimizes the potential interferences from other components and enables the simultaneous extraction and concentration of analytes . by far ,
most reported atpss are generated by mixing two solutions of polymers , such as dextran t500 and polyethylene glycol ( peg ) system , or by adding a high - concentration salt solution to an aqueous polymer solution .
the partition behavior of analytes between the polymer - rich phase and the aqueous phase can be controlled and optimized with a judicious choice of phase system . however , most of polymer - rich phase is highly viscous and opaque , rendering the follow - up analysis difficult . to address these issues ,
the development of a simple and environmentally friendly method with minimal use of volatile and toxic solvents used is of great importance .
ionic liquid ( il ) is a green solvent and a potential replacement for traditional volatile solvents . in 2003 ,
gutowski and coworkers first generated several atpss from different il and salts and determined the corresponding phase diagrams .
the identity of il is important for the separation / analysis of a specific analyte in il - based atps .
an optimal il - based apts should possess the following features : minimal emulsion formation , low viscosity , rapid phase separation , high extraction efficiency and low cost , and so forth . in this study
, we aim to develop a simple and  green  il - based atps extraction method with high efficiency and couple it with hplc for the analysis of ta and sy from food samples . for this purpose
, we selected 1-butyl-3-methylimidazolium bromide ( [ c4mim]br ) , which is not easy to emulsify and presents a lower viscidity compared with polymer - salt atps .
besides , [ c4mim]br was diffluent in water and methanol ( the hplc mobile phase ) .
analytes ( ta + sy ) could be extracted into il phase and analyzed by hplc with no further sample treatment .
we showed the success of this method in the separation / analysis of ta and sy from soft drink sample , candy sample , and instant powder drink .
all reagents were of analytical grade or higher in purity and purchased from sinopharm chemical reagent co. , ltd . ,
food yellow 4 ; 0.5  mg / ml ) , and sunset yellow ( sy ; c.i .
food yellow 3 ; 0.5  mg / ml ) were obtained from the national research center for certified reference materials ( beijing , china ) and were both prepared in distilled water to a final concentration of 100  g / ml . all glass containers were stored in 10% ( v / v ) nitric acid overnight and rinsed with distilled water before use . ils ( 1-alkyl-3-methylimidazolium bromide , [ cnmim]br , n = 4 , 6,8 ) were synthesized as described previously .
all spectral measurements were carried out using a model uv-2501 spectrophotometer ( shimadzu , japan ) .
phase separation of the sample solution was achieved with a centrifuge ( model tdl80 - 2b , shanghai anting science instrument factory , shanghai , china ) .
chromatographic analyses were performed on an agilent 1260 hplc system which consisted of a 1260 infinity quaternary pump with degasser gradient pump , a 1260 infinity variable wavelength detector , a 1260 infinity manual injector , and an open lab cds chemstation workstation ( agilent , usa ) .
a certain of [ cnmim]br was put into a 10.0  ml centrifugal tube . k2hpo4 solution or the other tested salt solution was added dropwise to the test tube until a turbidity formed , indicating the formation of a two - phase system .
thereafter , water was added dropwise to the test tube to obtain a clear one - phase system .
the bimodal curve was applied to characterize the phase diagram . 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 .
0.3  ml of [ c4mim]br and 1.0  ml of the mixed standard solution ( 100  g / ml sy + 100  g / ml ta ) or 1.03.0  ml of the sample solution were placed in a 10.0  ml centrifugal tube .
the mixture was diluted to 4.0  ml with distilled water and then 3.0  g k2hpo4 was added .
the tube was centrifuged for 2  min at 3500  rpm to ensure a complete phase separation .
20.0  l of [ c4mim]br phase was withdrawn using a microsyringe and injected into hplc for quantification .
chromatographic separation was achieved on a zorbax ods column ( 4.6  mm  150  mm  5m ) associated with a guard column packed with the same bonded phase .
the composition of the mobile phase at time zero ( the time of injection ) was 85% ammonium acetate ( 20  mm , ph 4.5 ) and 15% methanol . over the next 5  min ,
thereafter , the mobile phase was changed to ammonium acetate - methanol ( 2  :  98 ; v  :  v ) within 5  min
. finally , the chromatographic system was equilibrated during 5  min before the next injection . a flow rate of 0.8  ml / min was used throughout the 15  min run .
dual uv wavelength mode was used , with ta monitored at 420  nm and sy at 480  nm .
the mobile phase was filtered through 0.45  m micropore filter membrane prior to use .
the distribution behaviors of ta and sy between il phase and salt phase were characterized by the extraction efficiency ( e ) , partition coefficient ( k ) , and phase ratio ( r ) .
the parameters e , k , and r were defined as follows : 
 ( 1)e = cilvilcaqvaq+cilvil100%,k = cilcaq , r = vilvaq , 
 where cil and caq are the concentration of ta or sy in il phase and in salt phase , respectively , and vil and vaq are the volume of il phase and k2hpo4 phase , respectively .
the spectra and absorbance of ta or sy in [ c4mim]br / k2hpo4 system were determined as an example to study the distribution behavior and extraction efficiency . during each experiment ,
the il phase containing the analyte was collected to measure the uv - visible spectra on the uv-2501 uv - vis spectrophotometer .
the absorbance of ta and sy was measured at 420  nm and 480  nm , respectively .
the blank containing the same phase composition but without analyte was used as reference solution .
all samples , including soft drink , candy , and instant powdered drink , were obtained from a local market .
appropriate amounts ( 0.15  g ) of the samples were dissolved in 15  ml of water .
the carbonated drinks were degassed by ultrasonication for 5 minutes to remove the carbon dioxide .
a warming process ( 50c , 30  min ) was used for the complete dissolution of the sugar - based candy .
these solutions were centrifuged and the upper solutions were filtered through 0.45  m micropore filter membrane .
the filtrate was transferred to volumetric flask of 25.0  ml , volume adjusted to 25.0  ml using distilled water , and ready for il - atps extraction . the chinese national standard method ( gb / t 5009.35 - 2003 )
briefly , the sample solution was adjusted to ph 6.0 with 200  g / l citric acid solution and heated up to 60c .
then this solution was stirred with polyamide powder for 5  min and filtered through buchner filter using double - decked filter papers .
after filtering , the polyamide powder was washed with deionized water for 35 times , followed with the mixture of methanol - formic acid for 35 times .
the colorants absorbed by polyamide powder were eluted with 15  ml of eluting solution ( 30% ammonia solution mixed with ethanol in the volume atio of 3  :  7 ) , adjusted to ph 7.0 by acetic acid , and evaporated to near dryness , following which distilled water was added to the volume of 5.0  ml for hplc analysis .
in this paper the bimodal curve for the aqueous two phase systems of [ c4mim]br with a series of salts at 25c was investigated .
results showed that il - salt atps could be formed by adding appropriate amount of different salts , such as ( nh4)2so4 , na2so4 , and k2hpo4 .
other salts , such as k3po4 , kh2po4 , nacl , and nah2po4 , can not separate [ c4mim]br solution into two phases . as shown in figure 1(a ) , the phase separation capability of salts is k2hpo4 > ( nh4)2so4 > na2so4 , consistent with their solubility in water at 25c , suggesting a correlation between the phase separation capability of salts and their solubility in water .
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 - salt atps .
the bimodal curves determined at 25c for the il / k2hpo4 system were shown in figure 1(b ) .
when the weight percentage of k2hpo4 was more than 0.15% , the phase separation capability of il followed the order of [ c4mim]br  [ c6mim]br  [ c8mim]br < [ c10mim]br . in view of the lower synthetic cost and lower viscosity of [ c4mim]br than that of [ c10mim]br ,
the extraction efficiency mainly depends on the structure of analyte and its affinity towards the extractant ( i.e. , partitioning coefficient ) , phase ratio , and the number of extractions in the liquid - liquid extraction system .
it was also often used to estimate the migration ability of analyte between the two phases and the separation performance of extractant .
higher separation efficiency can be achieved with a greater partitioning coefficient k and a lower phase ratio r . in this paper
the effects of ionic liquid , salt , and temperature on extraction efficiency , partition coefficient , and phase ratio were assessed to obtain the optimal parameter .
the effect of ph on il - atps extraction ability of [ c4mim]br for ta and sy was studied in the ph range of 2.011.0 by the addition of britton - robinson buffer solution in the presence of 0.3  ml [ c4mim]br and 3.0  g k2hpo4 ( figure 2 ) .
it was foundthat the extraction efficiency remained relatively constant over the ph range and the ph has little effect on the extraction of ta and sy . with a fixed volume of [ c4mim]br at 0.3  ml ,
[ c4mim]br - k2hpo4 atps was formed when the amount of k2hpo4 was over 2.7  g in weight for ta and 2.9  g in weight for sy , respectively .
the influence of k2hpo4 concentration on the extraction efficiency , partition coefficient , and phase ratio was studied with 0.3  ml [ c4mim]br added and the results obtained were shown in figure 3 .
it revealed that the extraction efficiency obtained was above 95% with k2hpo4 of 3.003.40  g ( figure 3(a ) ) .
the partition coefficient of ta and sy was increased to a maximum value when the amount of k2hpo4 was set at 3.0  g and 3.1  g , respectively , then decreased with the increased amount of k2hpo4 .
the phase ratio of ta and sy was increased with the amount of k2hpo4 ( figure 3(b ) ) . in order to achieve quantitative extraction and higher separation efficiency ,
3.0  g of k2hpo4 was used in all following experiments for the simultaneous extraction of sy and ta .
the amount of [ c4mim]br used in the preconcentration procedure is a critical factor for obtaining a high extraction performance .
therefore , the extraction system was carefully studied to determine the lowest il - phase volume necessary for achieving the best extraction . in this system , the atps of [ c4mim]br - k2hpo4 could not be achieved with 3.2  g k2hpo4 if the volume of [ c4mim]br was less than 0.2  ml.so the effect of the volume of [ c4mim]br on extraction efficiency was studied in the range of 0.21.0  ml .
it was found that the extraction efficiency calculated was more than 95% in the concentration range of 0.31.0  ml ( figure 4(a ) ) . the partition coefficient of ta and sy got a maximum value when 0.3  ml [ c4mim]br was added , and then decreased with amount of il , whereas the phase ratio was increased with the increased amount of [ c4mim]br ( figure 4(b ) ) .
considering the extraction efficiency , enrichment factors , and the low consumption of [ c4mim]br , 0.3  ml [ c4mim]br il was used to achieve a higher extraction efficiency in the subsequent experiments .
the effect of temperature on the extraction efficiency and phase ratio of ta and sy by il - salt apts was also studied ( figure 5 ) .
the results revealed that the extraction efficiency of ta and sy was near to 100% within the entire tested temperature range from 10c to 70c .
the phase ratio was stable within this range and decreased when temperature was over 70c .
the curve of extraction efficiency and phase ratio of sunset yellow was superposed practically compared with that of tartrazine .
the reason was that higher temperatures result in more [ c4mim]br redissolved into k2hpo4-enriched bottom phase .
but the decrease of [ c4mim]br in top phase does not affect the extraction efficiencies of ta and sy .
this new extraction system can afford a wide temperature range for extraction of ta and sy . in the following experiments ,
the temperature was set at room temperature of 25c for the extraction of ta and sy .
the absorption spectra recorded for ta ( curve 1 and curve 2 ) and sy ( curve 3 and curve 4 ) before and after [ c4mim]br / k2hpo4 extraction , as scanned in the wavelength range of 350  nm700  nm against the reagent blank , respectively , were shown in figure 6 .
the spectra of these two colorants remained unaltered when they were extracted into the [ c4mim]br - rich top phase and practically superimposed before and after [ c4mim]br / k2hpo4 atps extraction .
this observation clearly indicated that no direct chemical ( bonding ) interactions were involved between colorants and ionic liquid in the il - atps . under the optimized conditions described above
, analytical parameters of the proposed hplc - uv - vis method such as linearity , sensitivity , correlation coefficients , limits of detection , and precisions were evaluated .
calibration graphs were constructed by plotting the peak area versus the concentration of analytes . for the tested ta and sy
, linearity was observed in the concentration range of 0.0150  g / ml . the other parameters are summarized in table 1 .
the results demonstrate that the method is sensitive for the determination of ta and sy in all studied samples .
the intraday precision of the proposed method was tested with 6 repeated injections of solution containing ta and sy standards at the concentration level of 0.5  g / ml . the relative standard deviations ( rsd )
the interday precision , expressed as rsd of the slopes of the calibration graphs obtained in three different days , was 5.6% .
due to the fact that the rsd is higher than 5% , the calibration graphs should be registered the same day as the real sample is analyzed . under the optimal conditions
, the proposed [ c4mim]br / k2hpo4 apts - hplc method has been adopted to determine ta and sy in candy samples .
typical chromatograms of ta and sy in the standard solution and candy sample were shown in figure 7 .
curves ( a ) and ( b ) were the chromatograms of the mixed standard solution ( ta + sy ) before and after il - salt atps extraction , respectively .
curves ( d ) and ( e ) were chromatograms of candy sample enriched by polyamide adsorption and il - salt atps , respectively .
figure 7 showed that ( 1 ) the standard solution ( curve ( a ) ) and real sample ( curve ( c ) ) have been enriched ( curves ( b ) and curve ( d ) ) by il - salt atps extraction ; ( 2 ) the response signal of ta and sy determined by il - salt atps extraction - hplc was consistent with gb / t 5009.35 - 2003 method ( curves ( d ) and ( e ) ) .
the proposed procedure has been applied to the determination of ta and sy in soft drink samples , candy samples , and instant powdered drink .
the recoveries of ta and sy in different food samples were in the range of 93.597.4% and 94.097.2% , respectively ( table 2 ) .
when compared with those achieved by the gb / t 5009.35 - 2003 method , as shown in table 2 , no significant difference was observed between the proposed method and gb / t 5009.35 - 2003 ( p > 0.05 ) .
a simple and rapid il - based atps consisting of [ c4mim]br and k2hpo4 coupled with hplc - uv was developed for the sensitive and simultaneous determination of sunset yellow and tartrazine in soft drink samples and candy samples . in this research , direct injection of the [ c4mim]br phase into hplc system for the quantification of ta and
this method proved to be efficient , simple and fast for separation / analysis of ta and sy in soft drink samples , candy samples and instant powdered drink .
 we proposed a simple and effective method , by coupling ionic liquid - based aqueous two - phase systems ( il - atpss ) with high performance liquid chromatography ( hplc ) , for the analysis of determining tartrazine and sunset yellow in food samples . under the optimized conditions , il - atpss generated an extraction efficiency of 99% for both analytes , which could then be directly analyzed by hplc without further treatment . 
 calibration plots were linear in the range of 0.0150.0  g / ml for both ta and sy . 
 the limits of detection were 5.2  ng / ml for ta and 6.9  ng / ml for sy . 
 this method proves successful for the separation / analysis of tartrazine and sunset yellow in soft drink sample , candy sample , and instant powder drink and leads to consistent results as obtained from the chinese national standard method . 
1. Introduction
2. Experimental
3. Results and Discussion
4. Conclusion