THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY NGUYEN HONG KIEN TOPIC TITLE: DEVELOPMENT OF AN EXTRACTION TECHNIQUE FOR DETERMINATION OF TRACE STRONIUM BACHELOR THESIS Study mode : Full-time Major : Environmental Science and Management Faculty : International Training and Development Center Batch : 2010-2015 Supervisor : Assoc Prof Do Thi Lan Prof Wu, Chien Hou Thai Nguyen, 15/01/2015 THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY INTERNATIONAL DEVELOPMENT AND TRAINING CENTER DEVELOPMENT OF AN EXTRACTION TECHNIQUE FOR DETERMINATION OF TRACE STRONIUM BACHELOR THESIS Student name : Nguyen Hong Kien Major : Environmental Science and Management Faculty : International Training and Development Batch : 2010-2015 Supervisor : Assoc Prof Do Thi Lan Prof Wu, Chien Hou Thai Nguyen - 2015 DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Degree Program Bachelor of Environment Science and Management Student name Nguyen Hong Kien Student ID DTN1053180064 Thesis title Supervisors Development of an extraction technique of determination of trace strontium Professor Wu, Chien-Hou and Assoc Professor Do Thi Lan Abstract: Strontium with its availability in environment and harmfulness to human health has pulled a lot of attention as well as the studies from researchers all around the world Even though there are a lot of extraction methods for substance traces in general and for strontium in particular, they still show their own drawbacks and strengths Therefore, it is very essential to develop more techniques for extraction with the improvements from the previous methods SPE and VALLME methods are two of new extraction methods which are under progress of development, which requires a lot of work in scientific field study This study delivers an overview picture for strontium toxicity which is currently got the low awareness from people Along with strontium toxicity, two extraction methods which are solid phase extraction (SPE) and vortex-assisted liquid-liquid microextraction method (VALLME) are presented with methodology, results and discussion The SPE-VALLME combined technique in order to maximize the efficiency from SPE and VALLME had also been experimented This study has contributed knowledge and confirmation to SPE and VALLME about its potential capacity in extraction and its reliability and accuracy for extraction method study, especially for strontium extraction in water sample or environmental sample i Keywords Strontium toxicity, extraction method, SPE, VALLME, chromatography Number of pages 44 pages Date of submission Jan 15th 2015 ii ACKNOWLEDGMENT First of all, I would like to express sincere thanks to the school board Thai Nguyen University of Agriculture and Forestry, Faculty of International Training and Development; Advanced Education program, thanks to the teachers who has imparted to me the knowledge and valuable experience during the process of learning and researching here This study was also supported and instructed by professor Do Thi Lan (Dean of Faculty of Environment, Thai Nguyen University of Agriculture and Forestry, Vietnam) and professor Wu, Chien-Hou (Department of Biomedical Engineering and Environmental Sciences, College of Nuclear Science, National Tsing Hua University, Hsinchu, Taiwan) I would like to give a special appreciation to Mr Wang Chin-Yi for helping me in time of doing this study I sincerely thank you! Taiwan, 2014 Student signature NGUYEN HONG KIEN iii TABLE OF CONTENT PART INTRODUCTION 1.1 RESEARCH RATIONALE 1.2 RESEARCH’S OBJECTIVES 1.3 RESEARCH QUESTIONS AND HYPOTHESES 1.4 STUDY SIGNIFICANCE 1.4.1 Scientific significance .5 1.4.2 Practical significance .5 PART LITERATURE REVIEW .7 2.1 STRONTIUM 2.1.1 Strontium related definitions 2.1.2 Strontium when entering the environment 2.1.3 Strontium impact on health 11 2.2 STRONTIUM EXTRACTION 13 2.2.1 IC (Ion Chromatography) machine .13 2.2.2 Research’s ion chromatography 16 2.2.3 Solid Phase Extraction (SPE) method 17 2.2.4 Vorted-Assisted Liquid-Liquid Mircoextraction (VALLME) 18 2.3 RESEARCH RELATED DOCUMENTS .19 PART METHODS 25 3.1 MATERIALS 25 3.2 EXPERIMENT 25 3.2.1 Solid Phase Extraction 25 3.2.2 Heating 28 3.2.3 Vortex-Assisted Liquid-liquid Microextraction 28 3.2.4 SPE-VALLME combined method 29 PART RESULTS .30 4.1 4.2 4.3 SOLID PHASE EXTRACTION (SPE) 30 VORTEX-ASSISTED LIQUID-LIQUID MICROEXTRATION (VALLME) 32 SPE-VALLME COMBINED METHOD 33 PART DISCUSSION AND CONCLUSION 35 5.1 DISCUSSION .35 5.1.1 Solid phase extraction (SPE) 35 5.1.2 Vortex-Assisted Liquid-liquid Microextration (VALLME) 36 5.1.3 SPE-VALLME combined method 38 5.2 CONCLUSION .40 REFERENCES 42 APPENDICES .44 iv LIST OF FIGURES Figure 2.1 Methyl Methanesulfonate 16 Figure 2.2 Crown ether of Sr resin 18 Figure 3.1 Spe method 26 Figure 3.2 Illustration of study experiment (SPE + VALLME) 29 figure 4.1: Elution curves of strontium with repeated condition (1ppm sr in 3m nitrate acid, 2ml of sample, 2ml of elution 30 figure 4.2 Recoveries of SPE processes with repetition (2 ml of 1ppm sr sample in 3m nitric acid, ml of elution) 31 figure 4.3 The improvement in signals from standard solution, solution with SPE applied and solution of SPE-VALLME combined method (100 ml of 20ppb strontium sample) 34 figure 5.1 Illustration of VALLME experiment data for reliability (experiment with 10ppb, 25ppb, 50ppb, 100ppb, 300ppb, 500ppb, 1ppm, 1.5ppm) 37 figure 5.2 The connection of SPE-VALLME combined method experiments’ results and reliability of the result (concentrations: 1ppb, 10ppb, 20ppb and 40ppb) 39 LIST OF TABLES Table 4.1 Data for SPE experiment with peak area, peak height, recovery (experiment with: ml of 1ppm Sr sample in 3M nitric acid, ml of elution) 32 Table 4.2 Data for VALLME experiment with concentration and peak area (experiment with 10ppb, 25ppb, 50ppb, 100ppb, 300ppb, 500ppb, 1ppm, 1.5ppm) 33 Table 4.3 Peak areas of standard and SPE-VALLME applied solutions with concentration of 1ppb, 10ppb, 20ppb, 40ppb 34 LIST OF ABBREVIATIONS DIW : Deioned water; SPE : Solid phase extraction; VALLME : Vortex-Assisted Liquid-liquid Microextraction PART INTRODUCTION 1.1 Research rationale Strontium extraction is rising as a hot topic in scientific study field Strontium has two types: the neutral and the radioactive Both of them have significant impacts to human health While the toxicity of strontium is still mysterious to human understanding due to its long-term impact for number of reasons, extraction method for strontium is very essential Currently, there are many extraction methods for strontium available, however each of them has its own strengths and weakness while the development of extraction method is never enough for scientific study, SPE and VALME could be a suggestion contributing to lists of extraction methods for not only strontium but for other substances (Chang W Y et al, 2012; Jakopic R and Benedik L, 2005) In addition, even Vietnamese people are not familiar with the strontium especially strontium toxicity due to low level of strontium in its natural condition, Vietnamese government is planning to build couple of nuclear power plants which would accidently release some radioactive fallouts- one of them could be strontium ninety With study of SPE and VALLME for strontium extraction, it could help to extract strontium ninety from environmental sample while with modification, SPE and VALLME are able to work not only with strontium but other substances or compounds 1.2 - Research’s objectives To study on the extraction methods for strontium which mostly focus on SPE, VALLME and the combination of those two methods By repeating experiments with SPE, the results of recovery varied in smaller range from 85% to 96% This was proportional to the real sample and higher volume of sample (same elution volume) according to Jakopic and Benedik’s study Figure VI would describe more about the repeatability of result for SPE experiment Figure 4.2 Recoveries of SPE processes with repetition (2 ml of 1ppm Sr sample in 3M nitric acid, ml of elution) 31 Belowing is the raw data for calculation of recovery for SPE experiment Table 4.1 Data for SPE experiment with peak area, peak height, recovery (experiment with: ml of 1ppm Sr sample in 3M nitric acid, ml of elution) Time of repetition Peak height Standard 26.9331 1.6485 3M_2ml 3.9251 0.23 14.57352 3M_4ml 20.3201 1.0961 75.44657 3M_6ml 0.3444 0.022 1.278724 3M_2ml 1.0583 0.0621 3.929366 3M_4ml 23.7033 0.949 88.00806 3M_6ml 1.1257 0.0695 4.179615 3M_2ml 1.9941 0.1126 7.403901 3M_4ml 21.4728 1.2322 79.72643 3M_6ml 0.4701 0.031 1.745436 3M_2ml 8.1744 15.3839 0.2033 0.5318 0.8818 0.0152 29.52176 55.55880 0.73421 3M_4ml 3M_6ml 4.2 Recovery (%) Peak area Total recovery achieved Note 91.29881 SPE only 96.11705 SPE only 88.87577 SPE only 85.81479 SPE only Vortex-Assisted Liquid-liquid Microextration (VALLME) For VALLME, because of low, varied concentration, recovery is not estimated correctly Parameters of results for VALLME experiment would be based on enrichment factor and repetition or reliability of VALLME method There is another way to test the reliability and repeatability of result for VALLME which is to repeat the experiment with different variables which in this case was concentration Concentrations delivered for this experiment were: 10ppb, 25ppb, 50ppb, 100ppb, 300ppb, 500ppb, 1ppm, 1.5ppm Due to the limitation of IC machine (unreliable of data when concentration lower than 20ppb), recovery was not the factor of concern in this experiment This below table shows the concentration and its peak area of signal collected by IC machine 32 Table 4.2 Data for VALLME experiment with concentration and peak area (experiment with 10ppb, 25ppb, 50ppb, 100ppb, 300ppb, 500ppb, 1ppm, 1.5ppm) Concentration Peak area Standard peak area 10ppb 1.0434 NC 25ppb 2.3794 NC 50ppb 5.8224 1.3371 100ppb 10.2997 NC 300ppb 27.7317 8.9923 500ppb 44.7046 NC 1ppm 91.4748 1.5ppm 143.7877 27.6894 42.552 4.3 Enrichment factor 4.354499 3.083938 3.303604 3.379106 SPE-VALLME combined method After experiments of SPE and VALLME, the SPE-VALLME combined method was further operated The result of experiment would be based on recovery, enrichment factor and repetition However because of low concentration of sample (120ppb), only repetition was concerned The difference in this experiment was instead of using 2ml of sample 1ppm, 100 ml of samples with various smaller concentration of strontium were applied The difference in signals of standard solution, solution with SPE applied and solution with SPE-VALLME combined method is described in figure 4.3 33 Conductivity (μS) Figure 4.3 The improvement in signals from standard solution, solution with SPE applied and solution of SPE-VALLME combined method (100 ml of 20ppb strontium sample) Due to low concentration of samples and unknown experiment error, table 4.3 would show the real peak area from standard solution and SPE-VALLME combined solution after combining SPE and VALLME based on the enrichment factor and recovery Table 4.3 Peak areas of standard and SPE-VALLME applied solutions with concentration of 1ppb, 10ppb, 20ppb, 40ppb Concentration (ppb) Peak area (μS) Enrichment factor Recovery Standard 0.0368 SPE-VALLME 2.0896 Not calculated Not calculated 10 0.3682 9.4295 Not calculated Not calculated 20 40 0.5213 1.6614 25.5361 40.6789 48.99 24.48 19.59% 9.79% 34 PART DISCUSSION AND CONCLUSION 5.1 Discussion 5.1.1 Solid phase extraction (SPE) Performance of 1ppm curve in figure 4.1 which is the dash line presents the standard solution The curve can be easily visualized that the curve got the highest peak and largest peak area, which means the amount of strontium in sample which remained in elution solvent was about 85%-97% Because of same proportion of sample adding, enrichment factor was not concerned in this experiment or enrichment factor was (no magnification in signal) One of differences between standard 1ppm solvent and others is that the 1ppm curve had limited in other signal due to the mixing of DIW and strontium only while the others processed in cartridge, which leads to get other substances, resulting in unwanted signals There is a small change in time set between 1ppm standard curve and others because the 1ppm standard was recalled from the other day which somehow the different in pressure (used for suppressor) could likely make this change However, it was the correction of time, the peak area and peak height were much dependent on the concentration and conductivity of analytes therefore the data was still reliable The recovery was calculated as the fomular: Recovery = x 100 As figure 4.2 witnessed, the recoveries of repetition were similar in range of 85%96% which were higher than 80% as in Jakopick and Benedik’s study (Jakopic R and Benedik L, 2005) Because of sensitive equipment of laboratory, the repetition for SPE experiment was four and could describe the reliability of experiment With average of 35 SPE recovery was 90.5% and no result below 80%, further experiment for SPE method could be conducted As in table 4.1, by detected the signal from minute five to six belonging to strontium signal, IC machine can easily combine with its attached software to deliver to user the peak area and peak height The data from table 4.1 was achieved from that The name 2ml, 4ml, 6ml means the volume of DIW added to cartridge, which would be up to 6ml As Jakopick and Benedik’s study, the signal would be much more on the 4ml of elution This was also an evidence for the reliability of their study and the contribution work to the extraction technique to trace strontium 6ml of elution was collected for totally signal detection for total recovery achieved 5.1.2 Vortex-Assisted Liquid-liquid Microextration (VALLME) As witnessed in table 4.2, there were a lot of concentrations had been tested for VALLME concentration As the table 4.2, peak areas are relevant to the concentration Up to the time of study, the enrichment factor was still getting to higher than while the theoretical enrichment factor was five Enrichment factor describes how large the signal of analyte has been magnified from the standard solution If enrichment factor is five, it means the signal from the original solution in case study has been magnified five times For instance, if the concentration of original solution is just 20ppb and the experiment gets enrichment factor of five, the signal of analyte will be equivalent or about the signal from the concentration of 100ppb Therefore, enrichment factor are very important to experiment for extraction method which includes theoretical enrichment factor and practical enrichment factor The theoretical enrichment factor was calculated by below formula 36 Theoretical enrichment factor = = times Even the practical enrichment factor not getting to 5, range from 3-4 still proves the increase in the concentration of sample, which helps the researchers in extraction strontium in laboratory sample or real sample By connecting the presentative points of concentration and peak area into a linear line, it is very convenient to witness the reliability of VALLME method on application to other concentration, which is described more in figure VII 160 140 120 100 80 60 40 20 y = 0.094x - 0.182 R² = 0.998 500 1000 1500 2000 Figure 5.1 Illustration of VALLME experiment data for reliability (experiment with 10ppb, 25ppb, 50ppb, 100ppb, 300ppb, 500ppb, 1ppm, 1.5ppm) As figure VII, the points are lined with R-squared very closed to R-squared is a statistical character measuring how close the data fitted together or in another term is coefficient of determination With achievement of R-squared as 0.9988, the results of VALLME experiment are very reliable and could be applied for other extraction method, not just for strontium extraction but for other substances With enrichment factor of three to four and R-squared of 0.9988 (indicating repetition or reliability), the result of VALLME experiment are very much succeeded, therefore further experiment or SPE-VALLME combined experiment could be conducted 37 5.1.3 SPE-VALLME combined method Figure 4.3 shows the huge difference in signal detection between standard solution, solution with SPE applied and SPE combined with VALLME solution With expectation of applying SPE combined with VALLME after good results from experiments of SPE and VALLME, 2ml of sample was changed 100 ml for familiar with the real situation in environmental sample (capability of large volume of sample, low concentration of analyte) Due to the change of sample volume, SPE method gave the theoretical enrichment factor which is calculated by formula Theoretical enrichment factor SPE = Sample of 20ppb strontium has very small signal as witnessed in figure VIII, however, with the large enrichment factor of 50 times as theoretical enrichment factor and 43-47 times (as recovery from 85-96%) as practical enrichment factor for SPE experiment, the signal of solution applied SPE had been magnified significantly The magnification in signal of strontium in ion chromatography has very important meaning, which makes the easiness in detection of strontium in environmental sample contaminated with unwanted substances As mentioned, the theoretical enrichment factor of VALLME was only five and was 3-4 as practical enrichment factor however when it combined with SPE, the enrichment factor could be reach as 250 times theoretically and as SPE experiment and VALLME experiment, the enrichment factor would range from 127 times (with 86% recovery for SPE and enrichment factor for VALLME) to 192 times (with 96% recovery for SPE and enrichment factor for VALLME) These are very big number which means the signal of strontium sample or strontium concentration would be magnify more than hundred times which helps a lot in study of strontium in environmental samples as well as detection strontium in water 38 As in table 4.3, for experiment of SPE-VALLME combined method, the signal of sample was magnified successfully Due to the low concentration of sample, enrichment factor and recovery of 1ppb and 10ppb was not calculated The signal of 20ppb and 40ppb was greatly decreased with unknown reason to 19.59% and 9.79% recovery for 20ppb and 40ppb, respectively The result did not reach the maximum magnification ability, theoritical enrichment factor or the pratical result with separated experiments but with the magnification of very low concentration of strontium, the signal was multiplized many times which helps to open the new way of study for extraction strontium and other substances Further study for SPE-VALLME combined method would need more investigating time to complete the method For reliability, with four done concentrations, the result is presented in below figure 45 y = 1.0163x + 1.3936 R² = 0.9763 40 35 Peak area 30 25 20 15 10 0 10 15 20 25 30 35 40 45 Concentration (ppb) Figure 5.2 The connection of SPE-VALLME combined method experiments’ results and reliability of the result (concentrations: 1ppb, 10ppb, 20ppb and 40ppb) 39 The points representing the data mostly lie on the linear function with R-squared as 0.9763 It was reasonably understandable to lower than the r-squared of VALLME experiment because with the combination of two different experiments, it would likely occur more variables, which makes the results more difficult to predict However with 0.9763 of r-squared, the result presents the good evidence for applying the method of SPE-VALLME combined method into study and extraction in real situation in environmental water sample 5.2 Conclusion SPE method has been approved again as very reliable technique with higher 80% of recovery and 43-47 times of enrichment factor if applying 100ml of sample and 2ml of elution VALLME with modification of Sr resin, the result is also appropriate and very much reliable for repetition with 3-4 times of enrichment factor (theoretically times) and 0.9988 r-squared Even though SPE-VALLME combined method showed very high r-squared as 0.9763, meaning very high reliability, the recovery and enrichment factor were decreased greatly due to unknown reason Therefore, further SPE-VALLME combined method needs further investigation and study to get the full promised potentials of this extraction technique Both SPE and VALLME are very good and new techniques for extraction of not only strontium but also other substances, which leads to its wide application in study as well as in real situation for environment samples With requirement of rapid time with high accuracy, SPE and VALLME support greatly to the extraction technique for determination of trace strontium in water sample and environmental samples By 40 applying crown ether to VALLME, this study also opens the new branch of study in extraction method in general and VALLME in particular Even with the limitation of time leading to not completely achieve the maximum potential application and extraction capability of SPE and VALLME, the study contributes a lot for acknowledge, scientific study and understanding both strontium and strontium extraction method 41 REFERENCES 1) Chang, W Y., Wang, C Y., Jan, J L., Lo, Y S., & Wu, C H (2012) Vortexassisted liquid-liquid microextraction coupled with derivatization for the fluorometric determination of aliphatic amines J Chromatogr A, 1248, 41-47 doi: 10.1016/j.chroma.2012.05.094; 2) Ojeda, C B., & Rojas, F S (2014) Vortex-Assisted Liquid-Liquid Microextraction (VALLME): Applications Chromatographia, 77(11-12), 745754 doi: DOI 10.1007/s10337-014-2669-x; 3) Kirrane, B M., Nelson, L S., & Hoffman, R S (2006) Massive strontium ferrite ingestion without acute toxicity Basic Clin Pharmacol Toxicol, 99(5), 358-359 doi: 10.1111/j.1742-7843.2006.pto_566.x; 4) L, J R a B (2005) Tracer Studies on Sr Resin and Determination of 90Sr in Environmental Samples Acta Chim Slov., 52, 297-302 5) McPherson, C A., Lawrence, G S., Elphick, J R., & Chapman, P M (2014) Development of a strontium chronic effects benchmark for aquatic life in freshwater Environ Toxicol Chem, 33(11), 2472-2478 doi: 10.1002/etc.2696; 6) Public health statement for strontium, Agency for toxic substances and disease registry, April 2014, US, accessed in www.atsdr.cdc.gov/ToxProfiles/tp159-c1b.pdf, January 1st 2015 7) Rezaee, M., Assadi, Y., Milani Hosseini, M R., Aghaee, E., Ahmadi, F., & Berijani, S (2006) Determination of organic compounds in water using 42 dispersive liquid-liquid microextraction J Chromatogr A, 1116(1-2), 1-9 doi: 10.1016/j.chroma.2006.03.007; 8) Yiantzi, E., Psillakis, E., Tyrovola, K., & Kalogerakis, N (2010) Vortexassisted liquid-liquid microextraction of octylphenol, nonylphenol and bisphenol-A Talanta, 80(5), 2057-2062 doi: 10.1016/j.talanta.2009.11.005; 43 APPENDICES Image 1: SPE experiment Image and 3: IC machine and column Image 4: Heating step Image 5: Centrigator and Experiment setting for centrifugtion Image 6: Vortex and timing process Image 7: Prof Wu and laboratory members (10/2014) 41 ... development of an extraction technique for determination of trace strontium, Jakopic and Benedik had defined the conditions and performance of tracers 20 in strontium extraction and its application... extraction technique of determination of trace strontium Professor Wu, Chien-Hou and Assoc Professor Do Thi Lan Abstract: Strontium with its availability in environment and harmfulness to human health... NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY INTERNATIONAL DEVELOPMENT AND TRAINING CENTER DEVELOPMENT OF AN EXTRACTION TECHNIQUE FOR DETERMINATION OF TRACE STRONIUM BACHELOR THESIS