High Performance Liquid Chromatography Application Possibilities to Determine Explosives

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1 High Performance Liquid Chromatography Application Possibilities to Determine Explosives JOSEF KELLNER 1), FRANTISEK BOZEK 1), ZBYNEK VECERA 2), VLADISLAV KAHLE 2), JIRI DVORAK 3), DANA MORAVCOVA 2) 1) Civil Protection Department, Faculty of Economics and Management, University of Defence 65 Kounicova, Brno, CZECH REPUBLIC 2) Institute of Analytical Chemistry of the Academy of Sciences of the Czech Republic, v.v.i. 97 Veveri, Brno, CZECH REPUBLIC 3) Language Training Centre, University of Defence 44 Kounicova, Brno, CZECH REPUBLIC Abstract: - In the paper it is presented the possibility of applying the high performance liquid chromatography (HPLC) to determine explosives. The implementation of HPLC on a functional sample of mobile continual analyzer of explosives is also presented as an example. Primary attention has been paid to the selection of a suitable column and the optimization of conditions for chromatographic analysis. It has been proven that the columns filled with surface-porous particles are suitable for being used in the analyzer of explosives. At the same time optimal experimental conditions have been discovered. Key-Words: - analyzer, columns, determination, explosives, liquid chromatography, porous particles, retention factor, separation factor, testing. 1 Introduction The necessity to increase the security of environment, critical infrastructure, including the individual and collective protection of military units and professionals, is in compliance with the tendencies followed in the NATO armies, in the NATO/SPS (Science for Peace and Security) projects, and in the EU countries. The implementation of measures increasing the security of units operating in peace and combat missions against the misuse of explosives and explosive devices by an enemy or in terrorist attacks against the systems and subsystems of critical infrastructure plays a significant role in risk reduction. The measures may be implemented either ex ante, or ex post". In both cases it is essential to use the means enabling us to identify explosives or their emissions and then determine the concentrations of corresponding volatile and semi-volatile substances. During work on the Defence research project it is expected to build an authentic analyzer of explosives on the microfluid basis enabling us to detect all organic nitrogen bound in explosives and explosive devices, including the inorganic compounds on the basis of nitrates (NO 3 - ) and nitrites (NO 2 - ). At the same time it is expected to develop a certified methodology to be implemented both in civil and military practice. 2 The Analysis of Current State Liquid chromatography, as opposed to gas chromatography, does not require sample to be transferred into a gaseous state. This fact enables liquid chromatography to analyze the substances that are thermally unstable or little volatile. Therefore such a technique is an ideal tool for the analysis of explosives. The high performance liquid chromatography (HPLC) is currently used for the separation of explosives mainly in forensic and environmental analyses. It is used in forensic analysis mainly for analyzing emissions after bursts and gunshots [1]. The on-line detection of explosives on the basis of HPLC for the security sphere has not been solved yet. Substances of various structures and nature have been used as explosives. Some of them are biodegradable and may become the basis of other substances of different nature, which are often toxic. The U.S. Environmental Protection Agency (U.S. EPA) recommends the HPLC for determining the explosives and their residues in the environmental elements. The EPA 8330A Method describes the way of determining 14 target analytes of selected explosives in water, soil and sediments, their degradation products and the substances used for their production (EPA 8330B describes even 17 analytes). The list of monitored substances is shown in Table 1 [2]. ISBN:

2 The methods describe the recommended way of treating the samples of water, soil and sediments, as well as the conditions of analysis through liquid chromatography. The UV-VIS detector is commonly used for determining nitroglycerine (NG) at the wavelength of 254 nm and for pentrite (PETN) at the wavelength of 210 nm. Methanol is applied as a mobile phase the most often. The column with C18 stationary phase is recommended. It is recommended to eliminate possible coelution of substances by using a comparative column with different selectivity (phenyl-hexyl or cyano-propyl phases). Table 1 Standard substances contained in EPA 8330A and EPA 8330B mixtures The substances written in italics have been used in the simplified mixture designed for optimizing the separation process Analyte Abbreviation CAS Number Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine HMX Hexahydro-1,3,5-trinitro-1,3,5-triazine RDX ,5-Trinitrobenzene 1,3,5-TNB ,3-Dinitrobenzene 1,3-DNB Methyl-2,4,6-trinitrophenylnitramine Tetryl Nitrobenzene NB ,4,6-Trinitrotoluene 2,4,6-TNT Amino-2,6-dinitrotoluene 4-Am-DNT Amino-4,6-dinitrotoluene 2-Am-DNT ,4-Dinitrotoluene 2,4-DNT ,6-Dinitrotoluene 2,6-DNT Nitrotoluene 2-NT Nitrotoluene 3-NT Nitrotoluene 4-NT A large number of further studies continue in this method. The range of determined substances remains the same, new methods of sampling are mainly tested and optimized, e.g. the application of solid phase microextraction (SPME) [3]. At the same time new types of detectors are developed. Great attention is paid to chemiluminiscence [4, 5], which seems to have good prospects when applied in case of the substances containing nitro groups (majority of explosives). A considerable number of papers deal with the analysis of explosives with the help of High Performance Liguid Chromatography with Mass Spectrometry detector (HPLC/MS) [6-10]. The separation and pre-concentration technique of liquid chromatography in the system with reversed phases has been selected as a suitable one for our purposes, i.e. the design and construction of mobile analyzer of explosives [11]. a) Methanol CHROMASOLV for HPLC, 99.9%, Sigma-Aldrich, Schnelldorf, Germany; b) Acetonitrile CHROMASOLV Plus, Sigma-Aldrich, Schnelldorf, Germany; c) Deionized water produced from distilled water with use of Ultra Clear UV system, SG Wasseraufbereitung und Regenerierstation GmbH, Hamburg, Germany; d) Uracil, Sigma-Aldrich, Schnelldorf, Germany; e) Standard compounds shown in Table 1, Sigma-Aldrich, Schnelldorf, Germany. 3.2 Tested Columns The following columns have been selected for testing: a) Poroshell 120 SB-C18, 2.7 μm, mm; b) Kinetex C18, 2.6 μm, mm; c) Zorbax SB-C18, 5 μm, mm. 3 Applied Chemicals, Methods and Devices 3.1 Applied Chemicals The following chemicals were used for testing the chromatography columns: 3.3 Devices AGILENT 1200 capillary system (Agilent Technologies, Palo Alto, CA) has been used for testing the columns. It included the following modules connected to each other: micro vacuum degasser, capillary pump, micro well plate autosampler, thermostated column compartment, diode array detector. ISBN:

3 The analyses have been carried out at the wavelength of 210 and 254 nm. The Chemstation for LC 3D system software has been used for operating the device, programming the individual sequences of analyses and data recording. 4 Outcomes and Discussion The requirements mainly for sufficiently effective separation in the shortest time have been considered in the selection of suitable columns. The conditions of separation have been optimized in the course of the project for the following columns: Poroshell 120 SB-C18, 2.7 μm, mm, Kinetex C18, 2.6 μm, mm and Zorbax SB-C18, 5 μm, mm. The mobile phases containing methanol and water in various ratios have been used for separating the analytes and characterizing the columns. The use of acetonitrile in the mixture with water as a mobile phase has been considered as an alternative. The above mentioned mixture has been simplified for optimization purposes and in order to meet the requirement for fast separation. The substances have been selected so that they covered a relatively broad range of values of distribution coefficients and included all the substances being primarily used as explosives (HMX, RDX, Tetryl, 2,4,6-TNT, 2,4-DNT, and 2,6-DNT). Pentrite, which is the main component of plastic explosives similar to Semtex, has also been added into the testing mixture. The aromates with one nitro-group do not have the capacity to detonate and that is why they were not included into the testing mixture. The exception is 4-nitrotoluene, which is approved as a marker. 4.1 Selection of a Suitable Column The optimum column for fast analysis of monitored analytes has to be sufficiently effective and at the same time it has to have prerequisites for maintaining its effectiveness even at high linear flow velocities of mobile phase through the column. Such a condition is met by the columns for Ultra Performance Liquid Chromatography (UPLC), which are filled with sub-2 μm particles. The main limitation to their application lies in a large hydraulic resistance to the flow of mobile phase through column, resulting in the necessity to have high pressures over 400 bars ensuring the stable flow of mobile phase. However, due to this fact it is excluded from its practical application in the given project. Relatively new particles with solid nuclei and porous layers (so-called surface-porous particles) represent a possible compromise. The columns filled with the above mentioned particles are currently available on a commercial basis. The sizes of particles used as sorbents are 2.6 μm, 2.7 μm and 5 μm. The Kinetex columns are filled with 2.6 μm particles by the Phenomenex company. These particles have solid nuclei 1.9 μm in diameter and the diffusion path is 0.35 μm long. The size of pore is about 90 Å. The particles 2.7 μm can be found in the Poroshell 120 (Agilent), HALO (MacMod Inc.) and Ascentis Express (Supelco) columns. These particles have solid nuclei 1.7 μm in diameter and the diffusion path is 0.5 μm long. The size of pore is about 120 Å. Motivation for using these types of sorbents for fast separation results from the short diffusion path enabling the substances to permeate between stagnant and mobile phases, high effectiveness of separation due to small diameter of particles, acceptable hydraulic resistance and a favourable course of van Deemter curves. The Poroshell 300 columns (Agilent) are filled with 5 μm particles. These particles have solid nuclei 4.5 μm in diameter and the diffusion path is 0.25 μm long. These columns are designated for separating large molecules, e.g. proteins. The reasons for using this type of sorbent are similar to the use of 2.6 and 2.7 μm particles. The 5 μm particles have much lower hydraulic resistance, though. The limitation factor for the separation of small substances (explosives) might be the size of pores of 300 Å Pressure Gradient of the Tested Columns The 50 % v/v methanol in water has been used for the testing and all the measurements have been carried out at the temperature of 25 C. The most favourable course of pressure gradient has been on the Zorbax SB-C18 column. Unfortunately, due to its low separation effectiveness the column has not been suitable for the fast separation of tested substances under isocratic conditions Comparison of Columns Analyzed under the Same Conditions The analyses have been carried out with the 50 % v/v methanol in water at the temperature of 25 C. The mean linear flow velocity of mobile phase through the column was 2.3 mm s -1, which corresponds with the flows of 250 μl min -1 for the Poroshell 120 SB-C18 and Kinetex C18 columns and the flow of 20 μl min -1 for the Zorbax SB-C18 column. The outcomes are clearly shown in Tables 2, 3, and Partial Conclusion It has been verified that the columns filled with surface-porous particles are much more effective for the monitored analyses than the standard 5 μm fully ISBN:

4 porous particles (Zorbax SB-C18 column), while the effectiveness of Poroshell 120 SB-C18 and Kinetex C18 columns in 50% v/v methanol is comparable. All the tested columns provide the selected analytes with the comparable retention factors k. Yet there are slight differences in the k values and when considering the requirement for fast analysis it is beneficial to apply the Poroshell 120 SB-C18 column. The most significant difference, up to k 1.0, has been monitored in case of pentrite as the most retained substance. The selectivity of all columns was similar with only small differences. The Poroshell 300 SB-C18, 5 μm, 1 75 mm column has not been tested in detail, because the required separation of explosive mixture has not been achieved even in 5% v/v methanol. Thus Poroshell 120 SB-C18 column may be recommended for further testing of the proposed system. Table 2 High Equivalent of Theoretical Plate HETP of used standard substances for the tested Poroshell 120 SB-C18, Kinetex C18 and Zorbax SB-C18 columns at the constant mean linear flow velocity of mobile phase through the column 2.3 mm s -1 Type of column HMX RDX Tetryl 2,4,6-TNT 2,4-DNT 4-NT PETN Poroshel Kinetex C Zorbax SB-C Table 3 Retention factor k of the used standard substances for the tested Poroshell 120 SB-C18, Kinetex C18 and Zorbax SB-C18 columns at the constant mean linear flow velocity of mobile phase through the column 2.3 mm s -1 Type of column HMX RDX Tetryl 2,4,6-TNT 2,4-DNT 4-NT PETN Poroshel Kinetex C Zorbax SB-C Table 4 Sepatation factor of the used standard substances for the tested Poroshell 120 SB-C18, Kinetex C18 and Zorbax SB-C18 columns at the constant mean linear flow velocity of mobile phase through the column 2.3 mm s -1 Type of column RDX/HMX HMX/Tetryl Tetryl/TNT TNT/2,4-DNT 2,4-DNT/4-NT 4-NT/PETN Poroshel Kinetex C Zorbax SB-C Optimizing the Conditions of Measurement Mobile Phase Composition The composition of mobile phase containing two components, namely methanol and water, has been optimized in order to reach the required separation of the individual components of standard sample during isocratic analysis. For drawing the dependence of the retention factor k on the content of organic component in the mobile phase there were several analyses carried out with the sample of standard mixture of explosives and the composition of mobile phase changed by 5 % v/v, ranging from % v/v of methanol in water. It results from the measurements that the mobile phase with 50% v/v of methanol in water is the most suitable for further tests, as the monitored analytes still have sufficient differences among their retention factors k Comparing the Effectiveness of Tested Columns The effectiveness of tested columns has been compared during the separation of selected explosives. The 50 % v/v methanol in water has been used for the testing and the testing mixture has been the simplified mixture of standard substances as it is described above. All the measurements have been carried out at the temperature of 25 C. After assessing the recorded data, van Deemter curves have been drawn showing the dependence of the Height Equivalent to a Theoretical Plate [µm] on the mean linear flow velocity of mobile phase through the u column [mm.s -1 ]. It results from the comparison of values that both columns, i.e. Poroshel 120 SB-C18 and Kinetex C 18, have comparable effectiveness Temperature Optimization Several feeds of explosive mixture have been carried out at the temperatures ranging from C ISBN:

5 in order to draw the dependence of the retention factor k on the temperature of column. This dependence has been measured on all the columns available. By increasing the temperature the time of analysis is reduced and with the increased flow 350 µl min -1 of the mobile phase through the Poroshell 120 SB-C18 column the monitored substances have been separated in less than 2 minutes Methanol Versus Acetonitrile The application of acetonitrile in mobile phase for the fast separation of monitored analytes have been considered with regard to its lower viscosity and low viscosity maximum, which would enable to use higher flows of mobile phase on the tested columns. However, worse selectivity and resolution for all the monitored analytes have been observed in acetonitrile. Hexogene and octogene have been a critical pair regarding separation. The high retention of pentrite seemed to be a critical factor in relation to fast separation. Although the mobile phases with methanol have higher viscosity compared to the mobile phases containing acetonitrile, there is better resolution and selectivity in case they are applied for the monitored analytes. Tetryl and 2,4,6-TNT is a critical pair for the separation in this system. There was a coelution of 2,4-DNT and 2,6-DNT on the tested column under the conditions mentioned above. 5 Conclusion It has been verified that the columns filled with surface-porous particles are much more effective for the monitored analytes in comparison with the standard 5 μm fully porous particles (Zorbax SB-C18 column). The effectiveness of Poroshell 120 SB-C18 and Kinetex C18 columns in 50% v/v methanol is comparable. All the tested columns show the comparable retention factors k with regard to the selected analytes. The conditions of isocratic HPLC separation for the Poroshell 120 SB-C18, Kinetex C18 and Zorbax SB-C18 columns have been optimized as well. With regard to the low effectiveness of separation on the Zorbax SB-C18 column, it is recommended to use Kinetex C18 and Poroshell 120 SB-C18 columns in the future as they have comparable effectiveness and selectivity for the monitored analytes. The Poroshell 120 SB-C18 column has been preferred due to the requirement for the shortest possible time of separation. The temperature of 60 C and the application of 50% v/v methanol in water as a mobile phase have been selected as suitable conditions for sufficiently fast and effective separation of monitored analytes. Acknowledgement The research has been carried out as part of the Defence Research Project, granted by the Ministry of Defence of the Czech Republic under No R/1. References [1] Yinon, J. Explosives. In Bousz, M. J. (Ed.), Handbook of Analytical Separations, 1 st Ed. Amsterdam: Elsevier, 2000, 742 pp. ISBN [2] U.S Environmental Protection Agency (U.S. EPA). Method 8330A. Nitroaromatics and Nitramines by High Performance Liquid Chromatography (HPLC), Revision 1, February [on line]. [ ]. URL: hazard/testmethods/sw846/pdfs/8330a.pdf. [3] Guarav, K. V., Malik, A. K, Rai, P. K. Development of New SPME-HPLC-UV Method for the Analysis of Nitro Explosives on Reverse Phase Amide Column and Application to Analysis of Aqueous Samples. J. Hazard. Mater, 2009, Vol. 172, No. 2-3, pp ISSN [4] Woltman, S. J. et al. Chromatographic Detection of Nitroaromatic and Nitramine Compounds by Electrochemical Reduction Combined with Photoluminescence Following Electron Transfer. Anal. Chem., 2000, Vol. 72, No. 20, pp ISSN [5] Tsaplev, Y. B. High-Performance Liquid Chromatography of Nitrate Esters with Chemiluminescence Detection. J. Anal. Chem., 2009, Vol. 64, No. 3, pp ISSN [6] Applied Biosystems. Trace Level Analysis of Explosives in Ground Water and Soil. [on line]. [ ]. Foster City: Applied Biosystems. URL: < oups/psm_marketing/documents/generaldocuments/ cms_ pdf >. [7] Bečanová, J., Friedl, Z., Šimek, Z. Identification and Deterimination of Trinitrotoluenes and Their Degradation Products Using Liquid Chromatography-Electrospray Ionization Mass Spectrometry. Int. J. Mass Spectrom., 2010, Vol. 291, No. 3, pp ISSN [8] Mathis, J. A., McCord, B. R. The Analysis of High Explosives by Liquid Chromatography/Electrospray Ionization Mass Spectrometry: Multiplexed Detection of Negative Ion Adducts. Rapid Commun. ISBN:

6 Mass Sp., 2005, Vol. 19, No. 2, pp ISSN [9] Yinon, J., Zhao, X. Identification of Nitrate Ester Explosives by Liquid Chromatography-Electrospray Ionization and Atmospheric Pressure Chemical Ionization Mass Spectrometry. J. Chromatogr. A, 2002, Vol. 977, No. 1, pp ISSN [10] Tachon, R. et al. Use of Porous Graphitic Carbon for the Analysis of Nitrate Ester, Nitramine and Nitroaromatic Explosives and By-Products by Liquid Chromatography-Atmospheric Pressure Chemical Ionisation-Mass Spectrometry. J. Chromatogr. A, 2007, Vol. 1154, No. 1-2, pp ISSN [11] Vecera, Z. et al. Wet Preconcentration Techniques for Real Time Determination of Gaseous Pollutants in Ambient Air. In Mladenov, V. et al. Proceedings of the International Conference on Development, Energy, Environment, Economics (DEEE 10). Puerto De La Cruz, Tenerife: WSEAS Press, 2010, pp ISSN ISBN:

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