Journal Aning of Chemical Purwaningsih, Technology Yanuardi and Metallurgy, Raharjo, Hendarta 52, 6, 2017, Agasi 1051-1055 APPLICATION OF IONIC LIQUID DISPERSIVE LIQUID- LIQUID MICROEXTRACTION FOR ANALYSIS OF N-NITROSODIPROPYLAMINE IN SALTED FISH Aning Purwaningsih, Yanuardi Raharjo, Hendarta Agasi Chemistry Department, Faculty of Science and Technology Universitas Airlangga, Surabaya, Indonesia E-mail: anp.unair@yahoo.com Received 05 January 2017 Accepted 20 July 2017 ABSTRACT An ionic liquid (IL) is a compound, composed of a cation, an anion and an alkyl, which has low volatility and good extraction properties. The combination of an ionic liquid and dispersive liquid-liquid microextraction with a GC-FID instrument, used for the determination of n-nitrosodipropylamine (NDPA), is an appropriate, accurate, easy and fast method. The following optimal parameters of the analysis have been established: ionic liquid [C 6 ], dispersing solvent volume 6 μl, 6 μl volume of solvent extract and sample volume 8 ml. The correlation coefficient of this method for concentrations from 2.0 to 10.0 g/l was 0.999. The detection limit for this method is 123 g/ml with an average accuracy of 95.02 % and coefficient of variation of 0.03 % - 2.38 %. The method is developed to detect NDPA in salted fish samples of 1.12 g/l. Keywords: ionic liquid, dispersive liquid-liquid microextraction, nitrosodipropylamine, salted fish. INTRODUCTION The solvent is a very important component in the process analysis. Solvents like the aliphatic hydrocarbons hexane and gasoline are widely used in the extraction of oil, tires, paint, aerosol carrier substance and disinfectants. Arene hydrocarbons as benzene, toluene and xylene, are used as solvents in the paint industry, for insecticides and agricultural chemicals, and alcohols are also widely used organic solvents. Generally, these solvents may cause many problems, since some of them are toxic, volatile, corrosive and carcinogenic [1]. Therefore, it is necessary to use solvents that can reduce the problems. The current trend is to develop ionic liquids as solvents. The advantages of an ionic liquid are its high capability as extractor of organic compounds and metal ions, insolubility in water, low volatility and non-flammability [2].. Based on these properties the ionic liquid has the potential to replace organic solvents for the preparation and analysis of a compound [3]. DLLME (Dispersive liquid-liquid microextraction) is a microextraction technique being developed by the researchers, because it has some advantages for sample preparation. DLLME is an easy, fast, economical process with a high recovery percentage, and a high concentration factor [3]. The combination between DLLME and an ionic liquid can be used in sample preparation for NDPA assay, contained in the food, being an accurate, safe, environmentally friendly method that does not generate a lot of waste and replacing organic solvents that are harmful. The aims for this paper is to introduce a new sample preparation for detection and analysis of carcinogenic compounds, especially nitrosamines, in diet. The production of the traditional salted fish generally employs fresh fish dipped into a solution of concentrated salt (NaCl), then dried in the sun. In addition to the use of salt, it is common to add potassium nitrite 1051
Journal of Chemical Technology and Metallurgy, 52, 6, 2017 and sodium nitrite as preservatives [4]. Compounds containing nitrite are precursors of nitrosamines, which are known as carcinogens. Nitrosamines can be formed in a reaction between nitrite and amine compounds in protein. The combination of an ionic liquid and DLLME with optimized ionic liquid type, volume of ionic liquid solution, volume of dispersing solvent and volume of NDPA are have been studied for NDPA analysis in salted fish, using a GC-FID instrument. EXPERIMENTAL Ionic Liquid-DLLME Method A 3 ml NDPA standard solution is put in a glass tube closed with a rubber cap. Six µl ionic liquid and 2 µl dispersant solvent (e.g. acetonitrile) are injected into the glass tube with a microsyringe, and centrifuged at 4500 rpm for 5 minutes. After the extraction process is complete, the ionic liquid, containing the analyte, is pulled back into the syringe (Fig. 1) and injected directly into the GC. Calibration curve without extraction A total of 5 types NDPA standard solutions with concentrations of respectively 2; 4; 6; 8; and 10 g/l were prepared and analyzed with GC to obtain chromatograms. The plots between area and concentration were used to obtain a calibration curve without extraction. Fig. 2. Diagram for the optimization of ionic liquid: a) [C 4 ]; b) [C 6 ]. RESULTS AND DISCUSSION Optimization of ionic liquid type In this study we used two types of ionic liquids, namely ([C 4 ]) and ([C 6 ]). Their selection was based on previous studies that extract a semi-polar target compound with a non-polar solvent.the longer chain results in a more nonpolar ionic liquid [2]. So it can be said that the ionic liquid ([C 6 ]) is more nonpolar than the ionic liquid ([C 4 ]). Based on the Fig. 2, it can be stated that the ionic liquid type ([C 6 ]) is the optimum ionic liquid and can be used in the next stage. Fig. 1. Scheme of the IL-DLLME procedure. 1052
Aning Purwaningsih, Yanuardi Raharjo, Hendarta Agasi Optimization of the Ionic Liquid Volume In the optimization of ionic liquid type, we used a concentration of 2 g/l NDPA, centrifugation speed of 4500 rpm for 5 minutes, 8 ml volume of the sample, ionic liquid ([C 6 ]) and dispersant solvent acetonitrile, with volume of 6 μl. The procedure was the same as the optimization of the ionic liquid volume (2, 4, 6, 8 and 10 μl). It can be seen in Fig. 3, that the optimum volume of the ionic liquid is 6 μl. From extraction results, it is observed that the increasing of the volume of the ionic liquid is followed by an increase in area generated, but when the volume is 8 ml, the area decreases, because the excess volume of ionic liquid leads to a decrease in the extraction efficiency. Higher volume of the ionic liquid, extracts more NDPA, but the increasing volume of ionic liquid causes decrease of the solubility in the dispersant solvent, so that a smaller area of NDPA is generated. These results are consistent with research conducted by Liu [5] and He [6], which show that the increasing volume of ionic liquid will increase the number of the extracted analyte, but at a certain amount the ionic liquid will saturate the analyte and the increase in the volume of ionic liquid decreases the concentration factor, so that the extraction efficiency is reduced. Therefore, the volume of 6 ml ionic liquid has been used for parameter optimization and subsequent analysis. Optimization of the Dispersant Solvent Volume The mixture of dispersant solvent with ionic liquid after introducing the target compound and the solvent generates a cloudy solution. This suggests that the role of the dispersant solvent is already underway, namely as a link between the solvents with different polarity. The dispersant solvents used in the optimization are ethanol, acetonitrile and acetone. Wherein the dispersant solvents of the three types represent the semi-polar nature solvent needed, while testing the capabilities of the dispersant solvents with different polarity. Based on previous research, we have concluded that acetonitrile is able to help the ionic liquid ([C 6 ]) to extract the NDPA compound well [7]. It is seen on Fig. 4 that for the methanol dispersant solvent, the amount of the extracted NDPA is very small. This is because the dispersant has a polarity which is almost the same as the solvent of the target compound, so that in both solutions, the ionic liquid and the target compound cannot function properly. Similar things happened in the solution of the acetone nonpolar dispersant. Because of its non-polar nature, acetone is not able to function as the solvent media for the ionic liquid and the target compound, because the ionic liquid has the same polarity as acetone. Thus, it can be concluded that the nature of the semi-polar acetonitrile as a dispersant solvent is appropriate and can be used in later stages. The extraction results for the optimization of the dispersant solvent volume can be seen in the Fig. 5. The excess volume of dispersant solvent causes a decrease in the extraction efficiency. This is because the excess volume of the dispersant solvent can cause the solution is not able to extract NDPA optimally. This shows that if the volume of ionic liquid is too much, then the amount Fig. 3. Ionic liquid volume optimization curve ([C 6 ]). Fig. 4. Optimization of dispersant solvent type. 1053
Journal of Chemical Technology and Metallurgy, 52, 6, 2017 Fig. 5. Optimization of dispersant solvent volume curve (Acetonitrile). of NDPA extracted is small. The results are consistent with research conducted by Liu [5] and He [6] which demonstrate that the increasing volume of ionic liquid will increase the amount of extracted analyte, but at a certain amount, the ionic liquid will saturate the analyte and the increase in volume of ionic liquid will make the concentration factor getting decreased, so that the extraction efficiency is reduced. Therefore, the volume of 6 ml ionic liquid has been used for parameter optimization and subsequent analysis. Optimization of the NDPA Volume After knowing the optimum volume of the dispersant solvent, we can proceed to the optimization of the volume of NDPA. As in the optimization of the volume of the dispersant solvent, the conditions remain the same in the optimization of the volume of dispersant solvent - Fig. 6. Volume optimization with NDPA. concentration of NDPA 2 g/l, centrifugation speed 4500 rpm for 5 minutes, volume of acetonitrile 2 μl, ionic liquid ([C 6 ]), volume of the Ionic liquid 6 ul. The procedure was the same as when in the optimization of the sample volumes (2, 4, 6, 8 and 10 ml). The extraction results for the optimization of the sample volumes can be seen in Fig. 6. Increasing the volume of the sample produces greater areas in the GC chromatogram, but if the volume of the sample is too high, the extraction efficiency is reduced. Enrichment Factor The Enrichment Factor shows the concentration that occurs during the extraction process, using IL-DLLME. The concentration of the analyte during the extraction process, using IL-DLLME, can be seen in Fig. 7. Fig. 7. Comparison between the NDPA extraction with IL-DLLME and without IL-DLLME. 1054
Aning Purwaningsih, Yanuardi Raharjo, Hendarta Agasi From this figure we calculated that the theoretical Enrichment Factor (EF th ) of the NDPA extraction process, using IL-DLLME, is 1200 times. While the real enrichment or True Enrichment Factor (EF tr ) is 1199.16 times. We can conclude that enrichment process using IL-DLLME is better, because the EF th result is close to the EF tr result. NDPA Analysis of Salted Fish Sample The analysis procedure for the NDPA concentration in salted fish has been similar to the procedure for the creation of the standard calibration curve of NDPA with the ionic liquid-dllme method. For the sample analysis, the constant sample volume was 8 ml, the ionic liquid used ([C 6 ]) was with volume of 6 μl, the dispersant solvent used was acetonitrile, with a volume of 6 μl, and the centrifugation was at 4500 rpm for 5 minutes. Measuring the NDPA concentration in the sample was done by using the average area from NDPA standard calibration curve, obtained with the ionic liquid-dllme method, as an Y-axis. The Equation of NDPA standard calibration curve with ionic liquid DLLME method is y = 10485x-223.5. The calculation with the experimentally obtained area showed that the concentration of NDPA in the sample was 1.12 g/l. CONCLUSIONS The combination of an ionic liquid and DLLME technique can be used in sample preparation for NDPA analysis by GC-FID. The optimal parameter values for this analysis are: ionic liquid ([C 6 ]) solution volume of 6μL, volume of the dispersant solution (acetonitrile) 6μL, and 8 ml sample volume. The studied combination gives good results when applied to the analysis of NDPA in salted fish with GC-FID. The NDPA concentration in the sample is determined to be 1.12 g/l. The detection limit for this method is 123 ppb with an accuracy range 100.04 % - 101.04 % and coefficient of variation up to 0.886 %. Acknowledgements The authors express gratitude to the Chemistry Department, Faculty of Science and Technology, and the Faculty of Pharmacy, Universitas Airlangga, for the research facility provided. REFERENCES 1. M.F. Kerton, Alternative Solvents for Green Chemistry, The Royal Societyof Chemistry, United Kingdom, 2011, p. 4-89. 2. M. Koel, C.H.Lochmuller, Ionic Liquid in Chemical Analysis: Practical and IndustrialApplications, 2 nd Analytical Chemistry Series CFC Duke University Press,13, 2009, 230-243. 3. M. Rezaee, Y. Assandi, M. Reza, E. Aghaee, F. Ahmadi, S. Berijani, Determination of organic compounds in water using dispersive liquid-liquid microextraction, Journal of Chromatography A, 1116, 2006, 1-9. 4. T. Margono, D. Suryati, S. Hartinah, Handbook of Food Technology, Information Center of Women in Development WWII-LIPI, Swiss Development Cooperation, 1993. 5. Y. Liu, E. Zhao, W. Zhu, H. Gao, Z. Zhou, Determination of four heterocyclic insecticides by ionic liquid dispersive liquid-liquid microextraction in water samples, J. Chrom. A, 1216, 2009, 885-891. 6. L. He, X. Luo, H. Xie, C. Wang, X. Jiang, K. Lu, Ionic liquid based dispersive liquid-liquid microextraction followed-high performance liquid chromatography for the determination of organophosphorus pesticides in water samples, Anal.Chim.Acta, 655, 1-2, 2009, 52-59. 7. A. Purwaningsih, Y.Raharjo, Applications of Microextraction Ionic Liquid-Based Green Chemistry for Analysis of Nitrosamines compounds, Report of Research, Department of Chemistry, Airlangga University, Surabaya, 2012. 1055