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1、 Analytical MethodsA rapid shaking-based ionic liquid dispersive liquid phase microextraction for the simultaneous determination of six synthetic food colourants in soft drinks, sugar-and gelatin-based confectionery by high-performance liquidchromatographyHao Wu a , Jing-bo Guo b , Li-ming Du a , ,

2、Hong Tian a , Cheng-xuan Hao a , Zhi-feng Wang b , Jie-yan Wang aa Analytical and Testing Center, Shanxi Normal University, Shanxi Linfen 041004, PR China bDepartment of Engineering, Shanxi Normal University, Shanxi Linfen 041004, PR Chinaa r t i c l e i n f o Article history:Received 8February 2012

3、Received in revised form 2March 2013Accepted 5March 2013Available online 14March 2013Keywords:Rapid shaking-based ionic liquid dispersive liquid phase microextraction Synthetic food colourantsHigh-performance liquid chromatographya b s t r a c tA novel and simple rapid shaking-based method of ionic

4、liquid dispersive liquid phase microextraction for the determination of six synthetic food colourants (Tartrazine,Amaranth, Sunset Yellow, Allura Red, Ponceau 4R, and Erythrosine in soft drinks, sugar-and gelatin-based confectionery was established. High-performance liquid chromatography coupled wit

5、h an ultraviolet detector was used for the determi-nations. The extraction procedure did not require a dispersive solvent, heat, ultrasonication, or additional chemical reagents. 1-Octyl-3-methylimidazolium tetrauoroborate (C8MIMBF4was dispersed in an aqueous sample solution as ne droplets by manual

6、 shaking, enabling the easier migration of analytes into the ionic liquid phase. Factors such as the C8MIMBF4volume, sample pH, extraction time, and centrifugation time were investigated. Under the optimum experimental conditions, the proposed method showed excellent detection sensitivity with limit

7、s of detection (signal-to-noiseratio =3 within 0.0150.32ng/mL.The method was also successfully used in analysing real food samples. Good spiked recoveries from 95.8%104.5%were obtained.2013Elsevier Ltd. All rights reserved.1. IntroductionColour is a main feature of foods. Its affect on people is not

8、 only visual; it is also associated with food variety, quality, and fresh-ness. Food colourants have been used to replace natural food col-our, which can be lost during preparation processes. Colourants are also used to prevent colour changes in the nal product (Berzas, Flores, Llerena, &Farinas, 19

9、99 and provide attractiveness to consumers, particularly children (Hofer &Jenewein, 1997. In recent years, natural food colourants isolated from suitable plants, fungi, or insects have been increasingly used. However, many natural colourants become unstable under processing conditions such as, light

10、, oxygen, and pH. Natural colourants are also more expensive than synthetic ones. The use of synthetic organic dyes has been recognised as the most reliable and economical method of restoring or providing colour to a processed product. However, some of these substances pose potential risks to human

11、health, especially when consumed in excess.To prevent indiscriminate use, laws and regulations based in toxicological studies on experimental animals and human clinical studies have been developed in many countries. The policies limit the types, purities, uses, and amounts of food colourants permitt

12、ed in food and drinks. Consequently, sensitive, accurate, and reliable methods for determining synthetic colourants are required to en-sure food safety.Several analytical techniques have been developed to facilitate the simultaneous determination of various synthetic food colourants. Such techniques

13、 include derivative spectrometry and other spectrophotometric methods related with chemometrics (Al-Degs, 2009; Berzas et al., 1999; Ni &Gong, 1997; Sayar &zdemir,1998, adsorptive voltammetry (Ni, Bai, &Jin, 1997, differential pulse polarography (Chanlon, Joly-Pottuz, Chatelut, Vittori, &Cretier, 20

14、05; Combeau, Chatelut, &Vittori, 2002, thin-layer chromatography (Morlock &Oellig, 2009, capillary electro-phoresis (Dossi et al., 2007; Ryvolova, Taborsky, Vrabel, Krasensky, &Preisler, 2007, high-performance liquid chromatography (HPLC(Minioti, Sakellariou, &Thomaidis, 2007; Pereira Alves, Brum, B

15、ranco de Andrade, &Pereira Netto, 2008; Vidotti, Costa, &Oli-veira, 2006; Yoshioka &Ichihashi, 2008, as well as ion chromatog-raphy (Chen, Mou, Hou, Riviello, &Ni, 1998.A recently proposed method, dispersive liquidliquid microex-traction (DLLME(Rezaee et al., 2006, is based on the formation of a tur

16、bid solution by the rapid injection of a mixture containingCorresponding author. Tel./fax:+863572057969.extraction and disperser solvents into an aqueous solution. The extraction solvent is dispersed into the aqueous sample as very ne droplets, enabling the analytes to transfer easily to the extrac-

17、tion solvent. When extraction equilibrium is achieved, phase separation is performed by centrifugation and the enriched ana-lytes in the sediment phase can be determined. Compared with other microextraction methods, this technique is more convenient, simple, and requires less expensive devices. More

18、 importantly, DLLME can be applied under batch conditions and extraction can be completed in several seconds, resulting in faster extraction and shorter analytical time.Room temperature ionic liquids (RTILsare a group of new or-ganic salts consisting of organic cations and various anions that are li

19、quid at room temperature. RTILs have been used as extraction solvents in place of organic solvents because of their unique phys-icochemical properties, such as negligible vapour pressure, misci-bility with water and organic solvents, good solubility in organic and inorganic compounds, and high therm

20、al stability as well as being environmentally benign (Pandey, 2006; Poole &Poole, 2010. DLLME based on ion liquids (ILs(IL-DLLMEwas introduced by Zhou et al. in 2008(Zhou, Bai, Xie, &Xiao, 2008. This approach needs an organic solvent as the dispersive solvent and heat to aid the complete dispersion

21、of a water-immiscible IL into the aqueous phase, then sedimentation by cooling with ice water. Extraction re-quires a specic heating time and cooling process, which is rela-tively time and energy consuming. To improve the extraction performance of temperature-controlled DLLME, ultrasound is used to

22、disperse the IL extraction solvent (Zhou, Zhang, &Xiao, 2009, but the cooling process and dispersive organic solvent are still needed to obtain a turbid solution. Then Yao and Anderson (2009reported a method for in situ IL formation DLLME, wherein the hydrophilic IL is completely dissolved in the aq

23、ueous phase and an ion-exchange reagent is added to form a water-immiscible IL. Although this method overcomes the weaknesses described above, the addition of excess ion-exchange reagent is required, which complicates the method.In the current work, a simple and efcient manual shaking-based method o

24、f IL-DLLME was developed. The procedure does not require a dispersive solvent, heat, ultrasonication, or additional chemical reagents, in contrast to conventional IL-DLLME. IL (C8MIMBF4was dispersed in an aqueous solution as ne drop-lets by manual shaking, promoting migration of the analytes to the

25、ionic liquid phase, then coupled with HPLC-ultraviolet (UVspec-trophotometry determination. The effects of various experimental parameters, including the C8MIMBF4volume, sample pH, extraction time, and centrifugation time, have been investigated and optimised for the extraction of six synthetic food

26、 colourants. 2. Materials and methods2.1. Reagents and standardsThe standard stock solutions of the colourants Tartrazine (TAR; C.I. Food Yellow 4; 0.5mg/mL,Amaranth (AMA;C.I. Food Red 9; 0.5mg/mL,Sunset Yellow (SUN;C.I. Food Yellow 3; 0.5mg/mL, Allura Red (ALL;C.I. Food Red 17; 1.0mg/mL,Ponceau 4R

27、(PON; C.I. Food Red 7; 0.5mg/mL,and Erythrosine (ERY;C.I. Food Red 14; 0.1mg/mLwere obtained from the National Research Center for Certied Reference Materials (Beijing,China. The mixed stan-dard solutions containing all colourants at 0.05mg/mLwas prepared by mixing and dilution of appropriate aliquo

28、ts from standard stock solution of each substance. Working solutions were prepared by appropriate dilutions of the mixed standard solutions with water. HPLC-grade methanol and acetonitrile were purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin,China. 1-Octyl-3-methylimidazoliu

29、m tetrauoroborate (C8MIMBF4, 1-hexyl-3-methylimidazolium chloride (C6MIMCl,and 1-oc-tyl-3-methylimidazolium chloride (C8MIMClwere obtained from Shanghai Cheng Jie Chemical Co., Ltd. (Shanghai,China. Milli-Q water (Millipore,Bedford, MA, USA was used throughout the study. All other reagents were anal

30、ytical grade and were pur-chased from Tianjin Kemiou Chemical Reagent Co., Ltd. (Tianjin, China. All solutions prepared for HPLC were ltered through 0.45l m membranes before use.2.2. InstrumentsThe chromatography equipment was a 1525binary HPLC pump and a 2489dual k UV detector from Waters (WatersCo

31、rporation, USA. The Waters Breeze software was used to control the instru-ments and acquire data. The chromatographic separation of the analytes was carried out on a Gemini C18column (5l m; 4.6mm 250mm; Phenomenex, Torrance, CA, USA. A pH meter (ModelpHS-3C, Shanghai Tianda Apparatus Co., Ltd., Chin

32、a was used for pH adjustment. A centrifuge Model TDZ4-WS (XiangYi Centrifuge Instrument Co., Ltd., China was employed to accelerate the phase-separation process.2.3. Preparation of the sample solutionAll samples, including soft drink, sugar-based and gelatin-based confectionery, were obtained from a

33、 local market. Appropriate amounts (0.32.5g of the samples were dissolved in 25mL of water. The carbonated drinks were degassed by ultrasonication for 5min. A warming process (50C, 30min was used for the complete dissolution of the sugar-based and gelatin-based confec-tionery. Samples were diluted t

34、o 50mL in a volumetric ask with an acetate buffer solution (0.2mol/L,pH 5.0. These solutions were ltered through a folded Xinhua paper lter (No.102, and the l-trate was collected after discarding the rst 15mL.2.4. Extraction procedureA homogeneous sample solution (10.0mL containing the ana-lytes was

35、 placed in a 15mL screw-cap conical-bottom graduated plastic centrifugal tube. Using a 500l L syringe, 350l L of RTIL was injected into the sample solution. Manual shaking (30times in 20s resulted in the formation of a turbid solution, which was centrifuged for 8min at a rate of 3500rpm (1685g . The

36、 upper aqueous solution was removed using a pipette, and the volume of residual IL was almost 180l L. Methanol was added to the IL res-idue enriched with analytes to obtain a volume of 300l L. Using a 25l L HPLC microsyringe, 10l L of the enriched solution was in-jected directly into the HPLC system

37、. All experiments were per-formed in triplicate. The syringe was rinsed with methanol and acetonitrile multiple times to remove residual analytes and IL. 2.5. Interference experimentsThe interferences were studied by analysing 10mL solution containing 100ng mL 1colourants and other chemical species

38、at different concentrations (0.1100l g mL 1, according to the rec-ommended extraction procedure. Tolerance limit of each species was taken as the largest amount yielding an error in the determi-nation of the analyte not exceeding 5%.2.6. Recovery and data handlingRecovery evaluations were performed

39、by spiking known amounts of the colours into the samples before processing and comparing the results with those from the same samples priorH. Wu et al. /Food Chemistry 141(2013182186183 spiking. Recoveries were estimatedtions and expressed as percentages.Final treatment of data and and recovery were

40、 performed using soft Excel.2.7. Chromatographic conditionsThe ow rate of the mobile min. The sample injection volume of the column was controlled at 30tained 0.1mol/Lammonium acetate justed by 10mol/Lsodium methanolacetonitrile (30:70,v/v.5%50%B (020min followed by detection wavelength was set at 4

41、30TAR and 510nm for the other grams of the post-extraction mixed tions are shown in Fig. 1. 3. Results and discussion 3.1. Comparison of ionic liquidIn the current study, ve MIMBF4,thanesulfonylimide(C6MIMNTfzolium hexauorophosphate (C6dazolium and 1-octyl-3-methylimidazolium MIMPF6were investigated

42、. A by manual shaking (Fig. 2a after The other commonly used ILs were DLLME method (Yao &Anderson, C6MIMCland C8MIMClwere also used. The ion-exchange re-agents were lithium bis(triuoromethanesulfonyimide(LiNTf2 and sodium hexauorophosphate (NaPF6. When 350l L of water-miscible IL and 0.35g of salt w

43、ere added to the aqueous phase, immiscible ILs C6MIMNTf2,C6MIMPF6,C8-MIMNTf2,and C8MIMPF6were formed. At the same time, a turbid solution with ne microdroplets was also formed, as shown in Fig. 2a. After 8min of centrifugation at 3500rpm, the IL phase was well separated from the aqueous phase, as sh

44、own in Fig. 2b.C8MIMBF4was found to have the best extraction efciency of the ve ILs for the articial colours. 3.2. Effect of the IL volumeThe amount of C8MIMBF4used in the preconcentration pro-cedure is a critical factor for obtaining a high extraction perfor-mance. Therefore, the extraction system

45、was carefully studied to determine the lowest IL-phase volume necessary for achieving the best extraction. The effect of C8MIMBF4was studied within the range of 250450l L. Fig. 3shows that with increased amount of C8MIMBF4,the peak area increased and reached a constant184 value when C8MIMBF4exceeded

46、 350l L, except for TAR. Further increases in IL reduced the enrichment factor achieved. Therefore, 350l L of C8MIMBF4was used in the subsequent experiments. 3.3. Effect of sample pHThe effects of pH on the extraction were studied within the pH range of 0.712using hydrochloric acid and sodium hydrox

47、ide, and the results are shown in Fig. 4. The extraction efciency of all col-ourants remained relatively constant over the pH range of 211. However, the extraction recovery decreased with further de-creased or increased pH. Considering most foods are weak acids or neutral, pH 5.0was used in all subs

48、equent experiments.3.4. Effect of extraction timeA turbid solution was easily formed at room temperature (251C; thus, the equilibration temperature in the extraction process was set at room temperature. Liquid-phase microextrac-tion is a time-dependent process. Consequently, the effect of the extrac

49、tion time was examined within the range of 020min at room temperature. In this experiment, extraction time was from the IL dispersion into the solution after manual shaking to before the initiation of centrifugation. The extraction time had no signi-cant effect on the extraction efciency. Therefore,

50、 to keep the anal-ysis time as short as possible, the cloudy solution was centrifuged immediately after the preparation at room temperature.In comparison with the other reported IL-DLLME methods, such as temperature-controlled (30min (Zhou et al., 2008, ultrasound-assisted (35min (Zhou et al., 2009

51、and in situ solvent formation (30s (Yao &Anderson, 2009 IL-DLLME, the rapid shaking-based IL-DLLME (20s has a signicantly shorter operation time for extraction.3.5. Effect of centrifuge timeCentrifugation, which controls phase separation, is a crucial step in the proposed method. The nal performance

52、 benets from a full phase separation. To achieve the best extraction efciency, centrifugation times within the range of 614min were examined at a rate of 3500rpm. At 8min, extraction recovery became con-stant, indicating the complete transfer of the IL phase. Therefore, the optimum centrifugation ti

53、me was determined as 8min. 3.6. Inuence of interfering substancesThe selectivity of the proposed method was studied using vari-ous chemical species that commonly interfere in the determination of colourants. The tolerance limit was dened as the concentration of a substance causing less than 5%relati

54、ve error for the six col-ourants using the proposed method. The samples contained a xed amount of colourants (100ng ml 1 and their effect was deter-mined using the proposed method, as described in Section 2.5. The results indicated that Na +, K +, NH 4+,Ca 2+, Zn 2+, Mg 2+, Mn 2+, Table 1Calibration

55、 equations, linear range, limits of detection, limits of quantication and coefcients of determination (R 2 of all colourants (n =11.Colourant Calibration equation (ng/mLLinear range (ng/mLDetection limit (ng/mLQuantication limit (ng/mLR 2Table 2Determination of colourants in food samples from the lo

56、cal market.Sample Colourant Concentration before extraction (ng/mLSpiked (ng/mLRecovery a (%Content of colourant (mg/kga Mean standard deviation (n =5.H. Wu et al. /Food Chemistry 141(2013182186185Br , Cl , I , SCN , SO 42, CO 32, HPO 42, PO 43, glucose, a -lactose, starch, dextrin, sucrose, citric

57、acid, and sodium cyclamate at 1000-fold, and Cu 2+, Co 2+, Cd 2+, Fe 3+, and F at 500-fold did not interfere with determination of the colours using the proposed method. This nding indicates the good selectivity of the method for determin-ing the studied colourants in food samples.3.7. Method evalua

58、tionThe calibration equations of the mixed standard solutions, coef-cients of determination (R 2, linear ranges, and limits of detection of all colourants are presented in Table 1. The calibration equations were calculated using the peak areas of the substances. Using the t-test, the slopes of the calibration equations of the mixed solutions were compared with those calculated from the measurements of individual standard solutions. No signicant differences were found. Quantication and detection limits were calculated follow-ing ACS guidelines (ACS, 1980, and were fo