Separation Of Radioisotope 113 m In Using Column Chromatography Based on Silica Gel Matrix

Radioisotope indium-113m ( 113m In) with half-life, T1/2 = 1.7 hours and gamma energy, E = 391 keV is suitable and meets the criteria as radiotracer in industry. 113m In radioisotope was obtained from tin-113 decay ( 113 Sn, T1 / 2 = 115 days) of 112 Sn (n,) 113 Sn neutron activation in nuclear reactor. The process of separation of radioisotope 113m In using column chromatography method based on silica gel matrix using 0.05 M HCI solution. Radionuclide and radiochemical purity tests were performed using the gamma-spectrometry method and paper chromatography. The final product specification in the form of 113m InCl3 is clear solution, pH 2, obtained yield of 81.83%, radionuclide purity of 90.22%, radiochemical purity of 91.61 ± 0,29% and stable for 3 days at room temperature.


INTRODUCTION
The rapid advance in radiotracer investigation has aroused widespread interest in the use of short-lived gamma-emitting radionuclides, especially for many industrial and nuclear medicine applications that require radioactivity for several minutes to several days.By using the short-lived radionuclide tracers, we could conduct measurement repetitions in short interval of time without having to worry about the remaining activities from the previous test.One of the disadvantages of using short halflife gamma emitter radionuclides as the industrial and nuclear medicine tracer is practical problems that only be used at near location to the nuclear reactor facility, because the short half-life limits the transport problem at the delivery distance.Therefore, timely availability in investigation location is the main obstacles that limit the use of this tracer in the industry and nuclear medicine.
To ensure a continuous supply of shortlived radionuclides, radioisotope generators (RG) are an effective strategy for solving practical problems.RG thus facilitates the activities in remote locations and also enables fast service delivery to handles urgent problems.Provision RG (in industry) would be cheaper than medical (nuclear medicine) radionuclide generators since there is no necessary to comply with strict regulatory requirements of radiopharmaceuticals.
Many couples of parent and daughter radionuclides have potential to be used in RG system, in this research, 113 Sn was selected to be evaluated on the basis of half-lives, types of radiation and energy as well as ease of production.Parent radionuclide, 113 Sn decay 98.2 percent to 113m In as a daughter radionuclide which possesses 391.69 keV of gamma energy and a half-life at 1.7 hours, likely to be used as RG 113 Sn/ 113m In in the nuclear medicine and industry.While the halflife of the parent is 115 days, this generator offers about 1 year of application (International Atomic Energy Agency, 2013;Mostafa, A. El-Sadek, El-Said, & El-Amir, 2009;Tárkányi et al., 2011).
Considering that 113 Sn radioisotope is neutron reaction products from 112 Sn (n, ) which has natural isotope abundances in the amount of 0.97 % and a very small crosssection of 1 barn, it is necessary to use 112 Sn enriched isotope (enrichment more than 95%). 113Sn radioisotope can be produced at low flux research reactors.Separation process 113m In via elution using diluted hydrochloride acid (theoretical: 70% of the activity 113m In and escaped 113 Sn < 0.01% of 113m In activity (Allan, Ali, Hanafi, & El-Azony, 2010).The chemical form of 113m In in RG 113 Sn/ 113m In eluate is In 3+ at low chloride concentration.While higher chloride concentrations, it formed complex chlorides such as InCl 2+ , and even InCl 2+ on a stronger chloride concentration converted to anionic form InCl 4-(Abdel-Halim, 2002;  Narita et al., 2014).
The utilization of radioisotopes 113m In that has a medium half-life (1.7 hours), low gamma energy (391 keV) and excess chemical properties of various concentrations of chloride, 113m In become the main choice in the application of nuclear techniques for industrial/ hydrology field.One of the activities in hydrology is to reduce the speed of sedimentation (silt/sediment) in boat harbor by knowing where it came from and the direction of movement of these sediments (International Atomic Energy Agency, 2011).Besides that,113m In has the potential utilization in various industrial fields as a tracer for aqueous phase, organic or solid.To understand the behavior of each stage of tracing process is important to investigate the performance of the system, indium-113m is possible for labeling complex compounds that stable in these phases (Toncheva, Gavazov, Lekova, Stojnova, & Dimitrov, 2011) 1000 mg Sn metal target containing quartz glass was placed in the aluminum nuclear grade inner capsule, and then closed by welding.Furthermore, the inner capsule was conducted via bubble method in water media.After that, the inner capsule was inserted into the outer capsule for irradiation process in a reactor RSG-GA Siwabessy (BATAN, Serpong) at CIP (Center Irradiation Position) with neutron flux of 1 x 10 14 n.cm -2 .s - for ± 94.5 hours.Irradiation was completed with Sn Safety Analysis Reports (SARs) document.
Irradiated material target was removed from the aluminum container, then put into a 250-ml beaker and dissolved in 100 ml of HCl 6 N. Dissolving process has been done on a heater with temperature of 80 °C, and was stirred gently at the same time with a magnetic stirrer to completely dissolved, then added 2 drops of concentrated H 2 O 2 .Thereafter, the solution was evaporated slowly until 50 mL left.Sn 2+ qualitative test was performed by pipeting 20 L of the solution diluted to 1 mL, and were added by 1 drop of ammonium molybdate.The change of blue color indicates cation Sn 2+ (Allan et al., 2010).In this process Sn was expected to be in the form of Sn 4+ cations.The solution was transferred into a glass vial and sealed using a cover vial glass (rubber cap).The whole work was conducted in hot cells.Activity measurement was done using spectrometry-γ MCA-HPGe at energy 225 keV ( 113 Sn) and 393 keV ( 113m In).

Solvent concentration variation
About ± 250 mg silica gel was weighted and placed into 5 vial bottle.Each vial is filled each vial with 1 mL solution of radioactive Snmetal (item 2.1) and set to be variation of solvent concentration of HCl (0.05; 0.1; 0.2; 0.5; 1 N) by adding distilled water.The solution was homogenized by stirring for ± 25 minutes.Then, silica gel was separated from the solution using a centrifuge.Next, pipette as ± 20μL filtrate and measured the radioactivity using Multi-Channel Analyzer instrument.Finally, do the calculation of the distribution coefficient.The distribution coefficient of three key radioisotopes i.e. 113 Sn, 113m In, and 117 mSn was calculated as the ratio of the radioactivity concentration of each radioisotope on solid matrix compared to the radioactivity concentration of those radioisotopes on the solution.

Stirring time variation
Weigh ± 250 mg silica gel into 6 vial bottle, fill each vial with 1 mL solution of active Sn-metal with concentration of HCL 0.1 N (item 2.2.1).The solution was homogenized by stirring for 5, 10, 15, 20, 25, and 30 minutes.Then, silica gel was separated from the solution using a centrifuge.Next, pipette as ± 20μL filtrate and measured the radioactivity using Multi-Channel Analyzer instrument.Finally, do the calculation of the distribution coefficient.

Weight variation of silica gel
Weigh silica gel into 6 vial bottle with different mass (100;200;250;300;400;500 mg), then fill each vial with 1 ml solution of active Sn-metal in HCl 0,1 N (item 2.2.1).The solution was homogenized by stirring for 25 minutes (item 2.2.2).Then, silica gel was separated from the solution using a centrifuge.Next, pipette as ± 20μL filtrate and measured the radioactivity using Multi-Channel Analyzer instrument.Finally, do the calculation of the distribution coefficient.

Separation process of 113m In by using silica gel chromatography column
Weigh ± 250 mg of silica gel into vial bottle then fill each vial with 1 mL solution of active Sn-metal in HCl 0,1 N (item 2.2.1).The solution was homogenized by stirring for 25 minutes (item 2.2.2).The solution was added to a glass column chromatography (ø 0.5 cm x 7.5 cm).Silica gel remains in the column, while the parent solution is removed and collected.113m In elution using 0.05 M HCl solution, the filtrate is collected 10 times of 1 mL per fraction/ vial.Then, 113m In radioactivity was measured by the Multi Channel Analyzer (HPGe-MCA).

Radionuclide and radiochemical purity test of 113m In.
Radionuclide purity was determined from gamma ray spectrum of 113m In as the HPGe-MCA analysis result.5 µL dosage of radionuclide 113m InCl 3 was dropped on filter paper, dried, put in a plastic wrapper, and counted for 60 minutes.γ-ray spectrum characteristic of 113m InCl 3 at 391 keV energy peaks.The radionuclide purity (KRN) of 113m In was calculated using the equation below: While the radiochemical purity was determined by paper chromatography using a mixture of 90% ethanol and 5 M HCl 10% (v / v) or acetone 85% as mobile phases and Whatman No.I chromatography paper as a stationary phase was observed at retention factor as 0.7 of In (III).113m InCl 3 solution was dropped at a distance of 2 cm from the bottom of Whatman 1, then the paper was put to the chamber that had been saturated by the eluent.Elution was carried out until the migration distance of the mobile phase reaches 14 cm.The paper was dried, cut every 1 cm and counted using γ-counter single channel analyzer (SCA).

RESULT AND DISCUSSION
Irradiation result of 112 Sn target 10 µL solution of the target (section 2.1) dropped on the vial and radioactivity concentration was measured using a spectrometry-γ MCA-HPGe.Gamma spectrum was analyzed, measurements were performed 3 times of repetition.Identification 113 Sn and 113m In radioisotopes were determined using BATAN BANDUNG NAA software (Lestiani, Muhayatun, & Adventini, 2009) and resulted in a graph between counts and the energy as shown in Figure 1.The peak at 255 keV was 113 Sn counts and 391 keV for 113m In radioisotope.Figure 1 showed the presence of other radionuclides than 113 Sn and 113m In which formed, were 117m Sn and 125 Sb radionuclides which derived from the core reaction of their isotopes by neutron at irradiation process.117m Sn came from the decay of irradiated result 116 Sn (natural isotopic abundance of 14 %), while 125 Sb resulted from the decay of irradiated 124 Sn. 116 Sn and 124 Sb isotopes were contained in metal Sn target (Cradarelli, 2008;Duyeh, Nana, & Titin, 2013).
Radioactivity 113 Sn as a form [ 113 SnCl 6 ] 2- obtained by measurement using the spectrometry-γ MCA-HPGe was 0.0467 mCi/mL, while theoretically at EOI (End Of Irradiation) should be 0.0504 mCi/mL, means obtained yield was 92.65%.Decay reactions that occur between parent radioisotopes ( 113 Sn) and daughter radioisotope ( 113m In) were secular equilibrium reaction where in 113 Sn has a half life 115 days, relatively longer than 113m In as 1.6 hours, allowed for separation by column chromatography using silica gel matrix (Saha, 2010).

Solvent concentration variation
Distribution coefficient values resulted from the experiment (show as Figure 2) indicated that the radionuclide 113 Sn and 113m In can be separated using silica gel in a suitable solvent.The higher value of KD indicated the nuclide had stronger interaction and well absorbed into silica gel matrix.The optimum concentration of HCl to impregnate 113 Sn as parent nuclide of 113m In was achieved in 0.1N where KD of 113 Sn was 156.27 ml/g.Although this process could not separate 113 Sn from other Sn radionuclide 117m Sn which had KD of 110.44 mL/g, impregnation process using this concentration can separated 113m In from its parent nuclide.On the optimum point (using HCl 0.1 N), 113m In had low KD.The KD of 113m In was around 20 mL/g which mean some 113m In was trapped in silica gel matrix.Higher KD value of 113 Sn and lower KD value of other nuclide indicated the better impregnation process.Beside solvent type and concentration, KD value is influenced by contact time and the amount of solid matrix.Figure 2 showed the optimum distribution coefficients was achieved in 0.1N HCl with KD of 113 Sn was 156.27 mL/g and KD of 113m In was 16.11 mL/g, therefore KD of 117m Sn was 110.44 mL/g.

Stirring time variation
The results of the analysis of the distribution coefficient determination based on agitation time as shown in Figure 3. Figure 3 visualized the optimum distribution coefficient was obtained at stirring process or contact time for 25 minutes with the values of KD of 113m In, 113 Sn and 117m Sn were 8.86 mL/g, 206.95 mL/g and 159.98 mL/g respectively.The effect of agitation time on KD was not clear enough.This might be caused from inconsistency of stirring speed.Generally, KD of 113 Sn is higher than 113m In almost in all variation of time.However, the KD value resulted from 25 minutes of agitation showed the highest KD of 113 Sn with the low KD of 113m In.This time parameter was used in preparation of the column chromatography.

Weight of silica gel variation
The analysis result of the distribution coefficient based on the weight variation of silica gel as shown in Figure 4. Figure 4 visualized the optimum distribution coefficient was obtained at 250 mg silica with the values of KD of 113 Sn was 240.12 mL/g, KD of 113m In was 8.79 mL/g, and KD of 117m Sn was 179.30 mL/g.Similar to time parameter determination result, the effect of the amount of solid matrix on KD of 113 Sn did not show the good pattern.Since the using of 250 mg of silica gel showed the optimum KD, this amount of solid matrix with agitation process within 25 minutes in 0.1 N of HCl were used in preparation stationary phase for separation process of 113m In from 113 Sn via column chromatography technique.In separation process using silica gel column chromatography 113m In and 113 Sn separation process was performed by the silica gel column chromatography system.The column was rinsed by 0.05 N HCl and the bottom of the column was sealed with a rubber stopper and aluminum cap (Figure 5a).To the 250 mg of silica gel was added 1 mL of 113 Sn in 0.1 N HCl. 113Sn solutions containing silica gel were homogenized by stirring for 25 minutes while heated at a temperature of 60 °C.Furthermore, the solution was added to a glass column (ø 0.5 cm x 7.5 cm) that the bottom of the column already contained activated carbon (Figure 5a), the aim of activated carbon addition was to absorb radionuclides 113 Sn and 117m Sn.Then the column was eluted by 0.05 N HCl so we get the final results of the elution profile of 113m InCl 3 solution (Figure 5b). Figure 5b showed there was no 113 Sn carried by HCl 0.05 N in all fraction.113m InCl 3 radioactivity measurements using spectrometer-γ MCA-HPGe was obtained of 0.0388 μCi/mL ( 117m Sn: 0.0034 μCi/mL), with the percent yield (separation) 81.83%.The most of 113m In was obtain in the first four fraction (4 mL elution) without 113 Sn being carried.This results show that the silica gel-based column chromatography combined with carbon active are potential to be used for separation of 113m In from irradiated natural tin.

Radionuclide purity (KRN)
113m InCl 3 radionuclide purity was tested using gamma ray (γ) spectroscopy, Multi-Channel Analyzer (MCA) equipment wiyh a gamma ray (γ) spectrum pattern as shown in Figure 6. Figure 6 indicates the characteristics of the γ-ray spectrum of 113m InCl 3 at 391 keV peak, which means that this production process can generate both 113m In products as 113m InCl 3 .
These results are relevant to the information described by Allan, et al. (2010), that the 113m In has gamma emission with an energy of 391 keV and a half-life of 1.7 hours.Data collection from gamma ray spectrum analysis 113m InCl 3 is summarized in Table 1.Table 1 showed 113m InCl 3 radionuclide purity was 90.22%, but there were other radionuclides, 125 Sb (0.47%) as the result of the core reaction of 124 Sn (n, ) 125 Sb, and 117m Sn radionuclides (7.90%) as the core reaction product 116 Sn (n, ) 117 Sn which has a natural isotopic abundance of 14% (Creech, Moynier, & Badullovich, 2017).InCl 3 radiochemical purity was determined by paper chromatography method by control the stationary phase compared to variable mobile phases.In the preparation of the solution of 113m InCl 3 , 113m In was expected to be in the form of a single compound, namely 113m InCl 3 .Paper chromatography systems were conducted in this study as summarized in Table 2. Table 2 showed the chromatographic system that was used is Whatman 1 chromatography paper as the stationary phase and a solution mixture of 90% ethanol: 10% HCl 5 N as the mobile phase with an elution time of 95 minutes.Using of Whatman 31ET chromatograph system as the stationary phase and a solution mixture of 90% ethanol: 10% HCl 5 N as the mobile phase with the elution time of 148 minutes also can be used.Chromatogram sample with this system is shown in Figure 7. Figure 7 showed the paper chromatography system which was used to determine the radiochemical purity 113m In are using Whatman 31ET paper as the stationary phase and solution mixture of 90% ethanol: 10% HCl 5 N as the mobile phase.In this study resulted in a chromatogram with Rf 0.7 to 0.8 as 113m In's peak with a radiochemical purity of 91.61 ± 0.29%.These results are relevant with study that used a mixture of 90% ethanol: 10% HCl as eluent with Rf 0.7 at Whatman 1 chromatography paper (Allan et al., 2010).

Stability determination 113m
InCl 3 solution stability in 3 days storage at room temperature showed that 113m InCl 3 remained clear and unchanged pH.While radiochemical stability was conducted by paper chromatography method by using mixtures of 90 % ethanol: 10 % HCl 5 N eluent.Radiochemical stability is showed in Figure 8. Figure 8 showed the results of stability test, 113m InCl 3 solution was stable and the purity was maintained at 90.96 ± 1.20% at room temperature.According to Uccelli et al., (2018), radiochemical purity of radiopharmaceutical, including radioisotope solution, could be affected by physico-chemical factor of the solution and environmental factor.Thus, by storing InCl 3 radioisotope solution in room temperature, it was sufficient to maintain radiochemical purity between 80% -100% (Uccelli et al., 2018).InCl 3 radioisotopes were obtained by irradiation of a metal tin target material (Snmetal) in the form of nature (Stano-112) by neutron activation at neutron flux of 1 × 10 14 n.cm -2 .s - for 94.5 hours.Characteristics of 113 Sn radioisotopes in 113 SnCl 3 chemical form were a clear solution, pH 2, the radioactive concentration of 0.0467 mCi/mL while theoretically was 0.0504 mCi/mL, obtained a yield of 92.65%.The optimum conditions of separation of radioisotopes 113 Sn / 113m In were obtained at a solvent concentration of HCl 0.1 N, stirred for 25 minutes and a silica gel's weight of 250 mg.The obtained 113m InCl 3 was a clear solution, pH 2, the activity was 0.0388 μCi/mL with a yield of 81.83%, radionuclide purity of 90.22%, and radiochemical purity of 91.61 ± 0.29%.

Figure 4 .Figure 6 .
Figure 4. Coefficients distribution at weight of silica gel variation

Table 1 .
Analysis result of 113m In gamma spectrum

Table 2 .
Paper chromatography system variation