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384 Chiang Mai J. Sci. 2010; 37(3) Chiang Mai J. Sci. 2010; 37(3) : 384-396 www.science.cmu.ac.th/journal-science/josci.html Contributed Paper Retention Behavior of Aromatic Amines with Some Ionic Liquids Mobile Phase in Semi-micro HPLC Yuppadee Nusai*, Hitoshi Koizumi, Masaki Tachibana, Kazue Tani, and Nobutoshi Kiba Department of Applied Chemistry, Faculty of Engineering, University of Yamanashi, Yamanashi, 400-8510, Japan. *Author for correspondence; e-mail: [email protected] Received: 5 January 2010 Accepted: 3 May 2010 ABSTRACT This research was aimed at investigating the retention behavior of aromatic amines with some ionic liquid mobile phases in semi-micro HPLC to explain the unclear separation mechanism. Aromatic amines (m-aminophenol, benzylamine, N,N-dimethylaniline, p-aminobenzoic acid, p-aminophenol, aniline, p-toluidine and N-methylaniline) were examined. The retention behavior of aromatic amines is greatly affected by the column equilibration time, ionic liquid concentration, mobile phase pH and alkyl chain length on the imidazolium cation of the ionic liquid. Semi-micro HPLC was used to increase separation power and sensitivity, and in addition to save on the mobile phase of the expensive ionic liquid. The separation mechanism involves ionic and hydrophobic interactions. Keywords: Ionic liquid; mobile phase; semi-micro HPLC; aromatic amine. 1. INTRODUCTION Ionic liquids (ILs) are a type of salts that o are liquid at low temperature (<100 C)[1]. ILs are normally composed of relatively large organic cations and inorganic or organic anions. They are polar solvents, environmentally harmless, nonvolatile, and nonflammable. Furthermore, it is possible to design solvents for specific applications by varying the lengths and branching of alkyl chains of the anionic core and the cationic precursor [2]. As a result of these ILs properties, ILs have been widely used in various chemical fields such as liquidliquid extraction [3-5], capillary electrophoresis (CE) [6-8]. ILs have been used increasingly to replace organic solvents for increasing the safety of workers in chemical laboratories and to decrease pollution in the environment. Recently, aqueous solutions of ILs have been used as mobile phases in conventional HPLC [2, 9-10]. However, the ionic liquids price is still high. Semi-micro HPLC is an interesting technique because a lower amount of solvent and a lower volume of sample injection are used (0.5 - 2 μL) and better sensitivity is achieved than with conventional HPLC. The use of the miniature analytical column reduces the expensive ILs solutions and simultaneously the waste liquid pollution in the environment [11]. Because a few of eight aromatic amines were coeluted, it was difficult to determine the retention factor. Therefore, we divided the Chiang Mai J. Sci. 2010; 37(3) aromatic amines into two groups as follows. The first group consisted of aniline (An), N-methylaniline (N-MA), N, N-dimethylaniline (N, N-DMA) and benzylamine (BA). The second group consisted of p-aminobenzoic 385 acid (p-ABA), m-aminophenol (m-Aph), p-toluidine (p-To) and p-aminophenol (p-Aph). The chemical structure and pKa value of these aromatic amines are shown in Figure 1. Figure 1. The chemical structure of analytes. (a) aniline(An); pKa = 4.70, (b) N-methylaniline (N-MA); pKa = 4.85, (c) N, N-dimethylaniline(N,N-DMA) ; pKa = 5.15, (d) benzylamine(BA); pKa = 9.33, (e) p-aminobenzoic acid(p-ABA) ; pKa1 = 4.65; pKa2 = 4.80, (f) m-aminophenol (m-Aph); pKa1 = 4.37; pKa2 = 9.815, (g) p-toluidine(p-To); pKa = 5.10 (h) p-aminophenol (p-Aph); pKa1 = 5.48; pKa2 = 10.46. 1-Alkyl-3-methylimidazolium-type ionic liquids were used, namely, 1-ethyl3-methylimidazolium tetrafluoroborate ([EMIm][BF4]), 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF4]) and 1-hexyl3-methylimidazolium tetrafluoroborate ([HMIm][BF4]). The chemical structure of these ionic liquids is shown in Figure 2. 1-Alkyl3-methylimidazolium tetrafluoroborates have a maximum absorbance value at about 216 nm. Figure 2. The chemical structure of ionic liquids. R = C2H5 = [EMIm][BF4]; λmax = 216 nm, R = C4H9 = [BMIm][BF4]; λmax = 216 nm, R = C6H13 = [HMIm][BF4]; λmax = 216 nm. 386 Chiang Mai J. Sci. 2010; 37(3) In this study, aqueous solutions of ILs ([EMIm][BF4], [BMIm][BF4] and [HMIm][BF4]) were applied as mobile phases in semi-micro HPLC to increase separation power and sensitivity, and in addition to save on the expensive ILs. Furthermore, we investigated the separation mechanism with aromatic amines. Various factors such as column equilibration time, ionic liquid concentration, mobile phase pH and alkyl chain length on the imidazolium cation of the ionic liquid were studied. This study is the first report using some ionic liquids mobile phases in semimicro HPLC. EXPERIMENTAL 2.1 Apparatus and Semi-micro HPLC Analysis The semi-micro HPLC system was composed of a LC-10ADVP Pump (Shimadzu, Japan), a 7520 injector with a 0.5 μL sample loop (Rheodyne, USA), a L-5025 column oven (Hitachi, Japan), and an 875-UV/Vis detector with a 1 μL cell volume (Jasco, Japan). A Develosil guard column (ODS-HG-S; 5 μm silica particle size; 10 mm 1.5 mm i.d.) and a Develosil ODS-T-3 column (end capping; 3 μm silica particle size; 100 mm 2 mm i.d.; trifunctional (polymeric); 20% carbon content; 3.4 μmol/m2) were used (Nomura Chemicals, Japan). The chromatograms were recorded on a D-2500 Chromato-Integrator (Hitachi, Japan). All chromatograms were obtained by isocratic elution at 0.2 mL min-1 flow rate. A sample injection volume was 0.5 μL. UV detection at 254 nm was used in the whole study. The retention factor (k) was calculated using this formula. 2. k= (1) where t R is the retention time of the analyte and t0 is the retention time of the unretained compound(NH4Cl). Five replicate injections were made to determine the retention time, and average values were used to calculate the retention factor. All o experiments were performed at 25 C. 2.2 Reagents An, N-MA, N, N-DMA, BA, p-ABA, m-Aph, p-To and p-Aph were purchased from Tokyu Kasei (Japan). [EMImBF 4 ] and [BMImBF 4] were purchased from Merck (Germany). [HMImBF4] was purchased from Wako Pure Chemical (Japan). 2.3 Preparation of Mobile Phases and Standard Solutions 2.3.1 Mobile phases preparation The stock solutions of specified ionic liquids concentrations were prepared by dissolving individually the known amounts of ionic liquids with deionized (DI) water. The stock solutions were filtered through 0.45 μm nylon membrane filters and were stocked in o a refrigerator at 4 C. The stock solutions were prepared weekly to protect the bacteria’s growth. The working mobile phases were prepared by diluting the stock solution to the required concentrations with DI water, and then they were adjusted to the required pH values with 10% (v/v) hydrochloric acid or 0.1 M sodium hydroxide. They were freshly prepared before use. There was no buffer utilization because 10% (v/v) hydrochloric acid was used for adjusting the mobile phase pH value. 0.1 M sodium hydroxide was only used when the excessively low mobile phase pH value was adjusted. 2.3.2 Standard solution preparation The stock standard solutions of 10 mM analytes were prepared by dissolving individually the known amount of analytes with 0.2 - 1 mL 1 M hydrochloric acid, and then adjusting them to the required volume Chiang Mai J. Sci. 2010; 37(3) with pH 3.0 hydrochloric acid aqueous solutions. They were stocked in a refrigerator o at 4 C and were prepared monthly. The pH 3.0 hydrochloric acid aqueous solution was prepared by adding 10% (v/v) hydrochloric acid to DI water. 10% (v/v) hydrochloric acid was gradually added to DI water until the aqueous solution pH value equaled 3.0 by pH meter measure. The 1 mM mixed working standard solutions were prepared by diluting the 10 mM stock standard solutions with pH 3.0 hydrochloric acid aqueous solution. They were divided into two groups. The first group consisted of m-Aph, BA, N,N-DMA and p-ABA ( 1 mM mixed aromatic amine series 1 standard solution). The second group consisted of p-Aph, An, p-To and N-MA (1 mM mixed aromatic amine series 2 standard solution). They were freshly prepared daily to avoid potential errors from decomposition of the compounds prior to 387 analysis with semi- micro HPLC. 3. RESULTS AND DISCUSSION 3.1 Effect of Column Equilibration Time on the Retention Factor (k) of Aromatic Amines The column equilibration time is an important factor in HPLC analysis. If the column is not completely equilibrated, the retention factor of analytes does not provide a constant value. The experiment was performed by using 5 mM (0.10% (w/ v) or 0.99 g L-1) [EMIm][BF4] aqueous solution at pH 4.0 as the mobile phase, an ODS-T-3 column, and N-MA as a test sample. The relationship between the retention factor of N-MA and the equilibration time is shown in Figure 3. It was found that when the column equilibration time was gradually increased from 1 to 18 hours, the N-MA retention factor steadily decreased until stable at 15 hours. Accordingly, the column equilibration Figure 3. Effect of the column equilibration time on the retention factor (k) of N-MA. column: ODS-T-3(end capping; 3 μm; 2.0 mm i.d. 100 mm) ; chromatographic condition: mobile phase: 5 mM [EMIm][BF4] aqueous solution at pH 4.0; flow rate: 0.2 mLmin-1; UV detection: 254 nm; sample injection volume: 0.5 μL. 388 Chiang Mai J. Sci. 2010; 37(3) time of the ODS-T-3 column was selected at 15 hours throughout all experiments. The aspect of [EMIm][BF 4] adsorbed on the ODS-T-3 column is shown in Figure 4. We can explain that gradually increasing the column equilibration time makes ethyl and methyl groups on imidazolium cations gradually interact with octadecyl and methyl groups of the stationary phase. The adsorbed imidazolium cations layer is formed gradually (pseudo-positive stationary phase); therefore, the protonated N-MA molecule is gradually eluted by electrostatic repulsion. After the pseudo positive stationary phase is completely formed, the retention factor of N-MA is consequently constant. Figure 4. The aspect of [EMIm][BF4] adsorbed on ODS-T-3 column. 3.2 Effect of [EMIm][BF4] Concentration on the Retention Factor (k) of Aromatic Amines The preliminary study had shown that the concentration of the ionic liquid affects the retention behavior of the aromatic amines. A series of 0.0, 1.0, 2.5 and 5.0 mM (0.00, 0.02, 0.05, 0.10% (w/ v) or 0.00, 0.20, 0.49, 0.99 g L-1) [EMIm][BF4] aqueous solutions at pH 3.0 was examined as the mobile phase to separate the 1mM mixed aromatic amine series 1,2 standard solutions. The result is shown in Figure 5. It was found that when the aqueous mobile phase without adding the [EMIm] [BF4] at pH 3.0 (a1, a2) was used, there were three problems as follows. First, band tailing of all aromatic amines occurred. Second, pABA and N,N-DMA, p-To and N-MA could not be separated. Third, the N,N-DMA peak was very broad. On the other hand, when the aqueous mobile phase containing 1 mM (0.02 % (w/ v) or 0.20 g L-1) [EMIm][BF4] at pH 3.0 (b1,b2) was used, it was found firstly that band tailing of all aromatic amines was greatly improved and symmetrical peaks of all aromatic amines were obtained. This is due to the fact that each ethyl and methyl group of imidazolium cations interacts with the octadecyl and methyl groups of the stationary phase by hydrophobic interaction, respectively. This is a disability of the alkyl groups of the stationary phase, which leads to the decrease in the possibility of dispersion interaction between the analytes and alkyl groups of the stationary phase. Furthermore, ionic liquids can suppress residual silanols on the ODS stationary phase even though end capping type. Secondly, the N,N-DMA and p-ABA peaks could be separated. Even when p-To and N-MA peaks could not be separated, the peak shape of these two analytes was sharper. Thirdly, the retention time of N, N-DMA was shorter than the retention time of p-ABA, and the N, N-DMA peak was clearly more symmetrical. Chiang Mai J. Sci. 2010; 37(3) 389 Figure 5. Chromatograms of Ar-amines with aqueous mobile phases containing various [EMIm][BF4] concentrations at pH 3.0. (a1,a2) 0 mM, (b1,b2) 1.0 mM, (c1,c2) 2.5 mM, (d1,d2) 5 mM. Chromatographic conditions: column: ODS-T-3(end capping; 3 μm; 2.0 mm i.d. 100 mm); flow rate: 0.2 mLmin-1; UV detection : 254 nm; sample injection volume: 0.5 μL. Peaks : (1) m-Aph; (2) BA; (3) p-ABA; (4) N,N-DMA, (5) p-Aph, (6) An, (7) p-To and (8) N-MA. 390 Chiang Mai J. Sci. 2010; 37(3) Figure 6A and 6B show the effect of [EMIm][BF4] concentration in the aqueous mobile phase on the retention factor (k) of aromatic amines. We found that increasing the [EMIm][BF 4] concentration affected the retention factor of all aromatic amines. It drastically decreased at 1.0 mM, and then slightly decreased at 2.5 mM and 5 mM. We can explain that the addition of 1.0 mM [EMIm][BF4] to the aqueous mobile phase, which causes the interaction between each ethyl and methyl group on imidazolium cations and octadecyl and methyl groups of the stationary phase. The surface of the ODS stationary phase is a positive charge; therefore, the protonated aromatic amines are rapidly eluted. It results in a decrease in the retention factor of the aromatic amines. Afterwards, when the concentration of [EMIm][BF4] was increased A to 2.50 mM, the retention factor of all aromatic amines was constant because the formation of the adsorbed imidazolium cations layer (pseudo-positive stationary phase) is complete. Moreover, when the concentration of [EMIm][BF4] was further increased to 5 mM, the result was that the retention factor of all aromatic amines slightly decreased. We conclude that forming the adsorbed imidazolium cations layer is equivalent above 2.5 mM [EMIm][BF4] concentrations. Considering the pKa value order of aromatic amines is as follows: BA> p-Aph>N,N-DMA>p-To>N-MA>An> p-ABA>m-Aph, the obtained retention factor order from using 1.0, 2.5 and 5 mM [EMIm][BF4] aqueous solutions at pH 3.0 as mobile phase was p-Aph<m-Aph<An<BA <p-To=N-MA<N,N-DMA<p-ABA. From B Figure 6. Effect of [EMIm][BF4] concentration in aqueous mobile phase at pH 3.0 on the retention factor (k) of aromatic amines. Chiang Mai J. Sci. 2010; 37(3) the obtained retention factor of all aromatic amines, we explain that each ethyl and methyl group on imidazolium cations interacts with octadecyl and methyl groups of the stationary phase by hydrophobic interaction, respectively. It makes the stationary phase surface become the positive charge. When the aromatic amine molecules are protonated, they become the positive charge as well. Consequently, they are eluted by electrostatic repulsion. If the retention behavior mechanism is mainly dominant by electrostatic repulsion, the aromatic amine will be eluted in the order of the pKa value. However, the obtained retention factor of the aromatic amine was not in accordance with the pKa value order. This is because of the methyl group effect on N-MA, N,N-DMA and p-To. From the methyl group effect, there is important evidence that indicates that the stationary phase surface still has a hydrophobic part as well. Accordingly, the separation mechanism involves electrostatic repulsion and hydrophobic interaction. 391 3.3 Effect of Mobile Phase pH on the Retention Factor (k) of Aromatic Amines It is understood well that the retention of the ionizable compounds on the reversed phase column depends on the pH of the aqueous portion in the mobile phase. For this reason, 2.5 mM (0.05% (w/ v) or 0.49 g L-1) [EMIm][BF4] aqueous solutions at various pH values (3.0, 3.5 and 4.0) were examined as mobile phases. The HPLC condition was started at a pH value of 3.0 for the following reasons: (1) The usable pH range of the mobile phase for the ODS column using the silica as the support material is 2-8. If a mobile phase with a pH value below 2 is used, the siloxanes linkage will crack. Moreover, a mobile phase with a pH value more than 8 is used, the silica will be dissolved. (2) All analytes can be absolutely protonated to cation forms at a pH value of 3.0. Figure 7A and 7B show the effect of the mobile phase pH on the retention factor (k) of aromatic amines. It was found that when the mobile phase pH was increased from 3.0 A B Figure 7. Effect of mobile phase pH on the retention factor (k) of aromatic amines. 392 to 3.5, the retention factor of An, N-MA, N, N-DMA, m-Aph, p-To and p-ABA increased, except BA and p-Aph. This behavior can be explained as follows: these seven aromatic amines are weak base, with the exception of BA, which is strong base; consequently, they are completely protonated to cation form in acidic aqueous mobile phase (pH 3.0). When the pH of aqueous mobile phase was increased to pH 3.5, they can dissociate less to cation form; however, most of the aromatic amines are still cation form, with the exception of p-ABA, which is a zwitterion. It causes a decrease of electrostatic repulsion force between the protonated aromatic Chiang Mai J. Sci. 2010; 37(3) amines and the positive stationary phase surface. When the mobile phase pH was further increased to 4.0, the retention factor of An, N-MA, N,N-DMA, m-Aph and p-To increased. It is because they can decreasingly dissociate to cation form, which causes a decrease of electrostatic repulsion force. As for the retention factor of p-ABA, it slightly decreased because it is zwitterion. It was observed that the mobile phase pH did not affect the retention factor of BA and p-Aph, because both analytes are always protonated species under the examined pH value. The typical chromatograms are shown in Figure 8. Figure 8. Chromatograms of Ar-amines with aqueous mobile phases containing 2.5 mM [EMIm][BF4] at various pH values (a1,a2) 3.0, (b1,b2) 3.5, (c1,c2) 4.0. Chromatographic conditions were shown in Figure 5. Peaks: (1) m-Aph, (2) BA, (3) p-ABA, (4) N,N-DMA, (5) p-Aph, (6) An, (7) p-To and (8) N-MA. Chiang Mai J. Sci. 2010; 37(3) 3.4 Effect of Alkyl Chain Length on the Imidazolium Cation of Ionic Liquid on the Retention Factor (k) of Aromatic Amines Figure 9A and 9B show the effect of alkyl chain length on the imidazolium ring of ionic liquid on the retention factor (k) of aromatic amines. This factor was investigated by using 2.5 mM (0.05 % (w/ v) or 0.49g L-1) [EMIm][BF4], 2.5 mM (0.06 % (w/ v) or 0.57 g L-1) [BMIm][BF4 ] and 2.5 mM(0.06 % (w/ v) or 0.64 g L-1) [HMIm][BF4] aqueous solutions at pH 3.0 as the mobile phases. It was apparent that with the increase of length of the alkyl substituent of imidazolium cation from ethyl, butyl to hexyl, all retention factors of aromatic amines decreased greatly. The longer alkyl chain can interact with the octadecyl more than the shorter alkyl chain; it 393 makes the stationary phase surface become the positive charge more. It is the cause of increasing the repulsive force of the same positive charge between the stationary phase surface and the ionized aromatic amines. N,N-DMA is stronger base than N-MA. If the separation mechanism of aromatic amines is controlled by electrostatic repulsion, N,N-DMA will be eluted more firstly than N-MA. However, due to the fact that the retention factor of N,N-DMA is longer than N-MA, this separation mechanism has an effect on account of hydrophobic force. These indicated that increasing the alkyl chain length on the imidazolium cation of ionic liquid increases the hydrophobic force as well. The typical chromatograms are shown in Figure 10. A B Figure 9. Effect of alkyl chain length of ionic liquid on the retention factor (k) of aromatic amines. 394 Chiang Mai J. Sci. 2010; 37(3) Figure 10. Chromatograms of Ar-amines with aqueous mobile phases containing 2.5 mM various ionic liquids at pH 3.0. (a1,a2) [EMIm][BF 4], (b1,b2) [BMIm][BF 4], (c1,c2) [HMIm][BF4]. Chromatographic conditions were shown in Figure 5. Peaks: (1) m-Aph, (2) BA, (3) p-ABA, (4) N,N-DMA, (5) p-Aph, (6) An, (7) p-To and (8) N-MA. Chiang Mai J. Sci. 2010; 37(3) 3.5 The Separation Mechanism with Ionic Liquid Mobile Phase for Aromatic Amines According to the obtained results of retention behavior of aromatic amine; therefore, we suppose that the separation mechanism with ionic liquid mobile phase for aromatic amines depends on repulsion between the imidazolium cations which interact with the methyl and octadecyl groups of the octadecylsilica stationary phase surface by hydrophobic interaction and the protonated aromatic amines. When the stationary phase surface becomes more 395 positive with ILs by the concentration, pH and alkyl chain length, it causes the protonated aromatic amines to be primarily repulsed by ionic interaction. Furthermore, because of other parts of imidazolium cations molecules are still hydrophobic, the protonated N-MA and N,N-DMA are also eluted by hydrophobic interaction. Consequently, the separation mechanism involves ionic and hydrophobic interactions. The separation mechanism with ionic liquid mobile phase for aromatic amines is shown in Figure 11. Figure 11. The separation mechanism with ionic liquid mobile phase for aromatic amines. 4. CONCLUSION The investigation indicated that the ionic liquids are potential to solving problems of aromatic amines separation such as band tailing and band broadening, because ionic liquids can play a multiplicity of roles, such as blocking residual silanol groups, modifying the stationary phase, or acting as ion-pairing agents. Furthermore, increasing the ionic liquid concentration and the alkyl chain length of ionic liquid makes the retention factor of all aromatic amines decrease clearly. On the contrary, the increase of the mobile phase pH 396 Chiang Mai J. Sci. 2010; 37(3) causes the retention factor of most aromatic amines to increase. The separation mechanism depends on competition between the imidazolium cations and the protonated aromatic amines in interacting with the stationary phase. The imidazolium cations can better interact, and make the stationary phase surface becomes the positive charge. Therefore the protonated aromatic amines are repulsed by ionic interaction. In addition, because of another part of imidazolium cation molecule is still hydrophobic, the protonated aromatic amines are eluted by hydrophobic interaction as well. These results are extremely useful for applying this system to analyse other compounds in the future. [5] Carmichael A.J., Earle M.J., Holbrey J.D., McCor mac P.B. and Seddon K.R., The Heck reaction in ionic liquids: a multiphasic catalyst system, Org. Lett.,1999; 1: 997-1000. 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