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Introduction of title
The terms of “forensic science” cover those professions which are involved in the application of social and physical science to the criminal justice system. Forensic experts are expected to find out the details of method used, to substantiate the choice of the applied technique and to give their unbiased conclusions. Therefore, the methods applied in forensic laboratories should assure a very high level of reliability and must be subjected to extensive quality assurance and rigid quality control programs. The legal system is based on the belief that the legal process results in justice. This has come under some question in recent years. Of course, the forensic scientist cannot change scepticism and mistrust single-handedly. He or she can, however, contribute to restoring faith in the judicial processes by using science and technology in the search for facts in civil, criminal and regulatory matters.
However, there are various chromatographic techniques used in forensic science since long time ago. Chromatography is probably the most powerful and versatile technique available to the modern analyst. In a single step process it can separate a mixture into its individual components and simultaneously provide an quantitative estimate of each constituent. Chromatography is a separations method that relies on differences in partitioning behavior between a flowing mobile phase and a stationary phase to separate the components in a mixture.
Separation of two sample components in chromatography is based on their different distribution between two non-miscible phases. The one, the stationary phase, a liquid or solid, is fixed in the system. The other, the mobile phase, a fluid, is streaming through the chromatographic system. In gas chromatography the mobile phase is a gas, in liquid chromatography it is a liquid The mobile phase is forced through an immobile, immiscible stationary phase. The phases are chosen such that components of the sample have differing solubilities in each phase. A component which is quite soluble in the stationary phase will take longer to travel through it than a component which is not very soluble in the stationary phase but very soluble in the mobile phase. As a result of these differences in mobilities, sample components will become separated from each other as they travel through the stationary phase. Different distribution of the analytes between mobile and stationary phase results in different migration velocities.
As all chromatographic separations are carried out using a mobile and a stationary phase, the primary classification of chromatography is based on the physical nature of the mobile phase. The mobile phase can be a gas or a liquid which gives rise to the two basic forms of chromatography, namely, gas chromatography (GC) and liquid chromatography (LC). The stationary phase can also take two forms, solid and liquid, which provides two subgroups of GC and LC, namely; gas-solid chromatography (GSC) and gas-liquid chromatography (GLC), together with liquid solid chromatography (LSC) and liquid chromatography (LLC).
There are few common examples as below :
It applies to volatile organic compounds. The mobile phase is a gas and the stationary phase is usually a liquid on a solid support or sometimes a solid adsorbent.
- High-performance liquid chromatography (HPLC)
A variation of liquid chromatography that utilizes high-pressure pumps to increase the efficiency of the separation.
- Liquid chromatography (LC)
Used to separate analytes in solution including metal ions and organic compounds. The mobile phase is a solvent and the stationary phase is a liquid on a solid support, a solid, or an ion-exchange resin.
- Size-exclusion chromatography (SEC)
Also called gel-permeation chromatography (GPC), the mobile phase is a solvent and the stationary phase is a packing of porous particles.
- Thin-layer chromatography (TLC)
Gas chromatography (GC)
A simple and rapid method to monitor the extent of a reaction or to check the purity of organic compounds. The mobile phase is a solvent and the stationary phase is a solid adsorbent on a flat support.
Among all the chromatographic techniques, thin-layer chromatography is the easiest method to demonstrate in a classroom how chromatography is used to separate materials.
Case Study 1:
Extraction and analysis of flunitrazepam/7-aminoflunitrazepam in blood and urine by HPLC and GC-MS
Flunitrazepam is a drug which is famous used in the area of drug-facilitated sexual assaults (DFSA) (Madea et al., 2004). Flunitrazepam solution is odourless, tasteless and colorless. These features, together with its potential use with ethyl alcohol have often caused the drug to be used an incapacitating agent in rape or robbery. (Calhoun et al., 1996.; Ledray and Amerg, 1996 .; Anglin et al, 1997) It belongs to most potent hypnotic benzodiazepines and prescript in most European countries, in Australia, South Africa and Latin America. In US, flunitrazepam is not approved. Nevertheless, the drug is smuggled into the US and its metabolite has been identified several times in clinical samples during drug screening (Valentine et al., 1996; Woods et al., 1996; Saum and Inciardi, 1997). The drugs in its parent form has been administered orally and by intravenous injection in 0.5-2 mg. Previous published methods for the extraction of flunitrazepam and its metabolites have used both of liquid-liquid and solid-phase extraction (Robertion and Drummer, 1995).
Flunitrazepam undergoes reduction to 7-aminoflunitrazepam(7-AF) After acetlyation the metabolites are excreted in urine (Baselt and Cravey, 2004). 7-AF was the most abundant metabolites among others and could be found up to 72 hours after ingestion of 1mg of drug, whereas the parent drug was not detectable in urine (Salamone et al., 1997). Instrumental methods of analysis have used electron-capture detection gas chromatography (Cano et al.,1997), and without prior derivatization (deSilva and Bekersky, 1974), high performance liquid chromatography (Sumitrapura et al.,1982) with mass spectrometry (LeBeau et al.,2000). Gas chromatography-mass spectrometry (GC-MS) using chemical derivatives such as BSTFA (Salmone, 1997) and MTBSTFA (United Chemical Technologies).
All assay methods for flunitrazepam and its metabolites show some limitations. Benzodiazepine immunoassays often lack the sensitivity required to detect flunitrazepam metabolites in urine (Manchon et al., 1985; Rohrich et al., 1994; Huang and Moody, 1995). The on-line enzyme hydrolysis may improve the detectability of 7-AF with immunoassay (Drummer et al.,1993; Beck, 1997). Whole blood radioimmunoassay, specific for flunitrazepam, showed no crossreactivity for 7-AF (West et al., 1995). Gas chromatography (GC) with sensitive, but unspecific electron capture detection (ECD) allows detection of flunitrazepam, but not 7-AF. In contrast to GC, all substances involved may be separated by high-performance liquid chromatography (HPLC) without any derivatization. The weak points of HPLC with UV detection were: questionable specificity and low sensitivity (the limit of quantitation was 10 mg/l (Boukhabza et al.,1991; Guichard et al., 1993; Robertson and Drummer, 1995). The purpose of the present study is to identify a solution to problem of extraction, detection and quantification of this drug and its metabolite in whole blood and urine that other workers in the field have identified in that flunitrazepam is easily converted into metabolite(s)
Calibrators and controls
Stock solutions of flunitrazepam, 7-aminoflunitrazepam and nitrazepam (10µgmL-1) were made up separately in 10mL volumetric flasks. This was performed by the addition 100µL of each solution (1mgmL-1) to distilled water and making up to the mark with the same. Working solutions (1000ngmL-1) of each drug were made by serial dilution in separate 10mL volumetric flasks.
Whole blood or urine
Nitrazepam (IS) (100µL of 1000ngmL-1) was added to samples of human drug free whole blood/urine (1mL) in 16mm-125mm screw top test tubes. Drug free bovine blood and post mortem human urine was employed. Both sources of blood had previously treated with sodium fluoride. To control stability of the drugs, the analytes were added to the blood/urine at the point of analysis (as storage may lead to conversion of the parent to the metabolite (Robertson and Drummer, 1998). For the calibrators, to each sample was added a known amount of flunitrazepam and 7-aminoflunitrazepam (0, 10, 25, 50 and 100ng). In the case of the controls, in addition to the nitrazepam (100ng), flunitrazepam and 7-aminoflunitrazepam were added at a level of 40ngmL-1 . All determinations (calibrators and controls) were carried out in duplicate. The tubes were vortex mixed (approximately 1min). To each sample was added 10mL of distilled water. The tubes were again vortex mixed for approximately 1min. The tubes were centrifuged at 2000-g for 10min. The supernatant liquid was applied to previously conditioned solid phase columns.
The SPE columns were placed in numbered slots in the manifold. Each column was conditioned with 1- 3mL of methanol followed by 1- 3mL of distilled water. These were allowed to percolate through the sorbent under gravity. The level of distilled water was held just above the solid phase sorbent bed to prevent it from drying out.
The supernatants were loaded onto the conditioned solid phase sorbent and allowed to pass through with the aid of gravity. After the samples were drawn through the solid phase columns, the sorbent was washed with 1- 3mL of distilled water, this was followed by 1- 3mL of 1M of acetic acid. The columns were dried under full vacuum for 5min. The cartridges were then washed with 1- 3mL of hexane after which they were dried under full vacuum for 5min.
Elution process prior to high performance liquid chromatography (HPLC)
In liquid chromatography, the eluant is the liquid solvent. The drugs and internal standard were eluted from the columns using an organic solvent mixture consisting of ethyl acetate/methanol (80:20). The volume of elution solvent employed was 2- 3mL. The eluants were collected in screw top test tubes (10mL capacity) at a rate of approximately 1mLmin-1. The combined extracts were evaporated to dryness under a gentle stream of nitrogen at 40°C. The residue was dissolved in 0.1% (v/v) aqueous TFA (100µL). This solution was transferred to an auto sampler vial containing a low volume insert (200µL) for analysis by HPLC.
Process employed prior to gas chromatography-mass spectrometry
The residue was dissolved in ethyl acetate (25µL) and 25µL of Pentafluoropropionic acid (PFPA) was added as the derivatizing reagent after the collection and evaporation of the combined eluants. Next, this solution was heated at 80°C for 30min. After cooling, the PFPA was evaporated to dryness at 40°C under a gentle stream of nitrogen. The residue was dissolved in 50µL of ethyl acetate. This was transferred to an autosampler vial containing a low volume insert (200µL) for analysis by GC-MS.
Altough there are low levels of flunitrazepam and 7-aminoflunitrazepam from whole blood/urine, the use of butyl endcapped solid phase columns give an selective solution to the problem of extracting them. The extraction relies on the hydrophobic interaction of the sorbent and the analyte solution. By applying a shorter alkyl chain, example C4 bonded to a silica structure, the polarity of the solid phase sorbent is increased. This works to aid the process of sorbing the drugs onto the solid phase column.
In the literature, the pKa values of flunitrazepam and nitrazepam are reported as 1.8 and 3.2 respectively (Moffat et al., 2004). Information relating to the pKa of 7-aminoflunitrazepam is not easily available. From this data, it is a natural assumption that all three analytes are acidic in nature. The pH modification of the blood and urine samples by distilled water make it easier to extract the drugs from the bio-fluids efficiently. It also assists the smooth flow of the solution through the column.
The use of liquid chromatography with photodiode array (LC-PDA) shows the ability of this system to separate and detect these drugs at low levels. It also expands the possibility of opening up this technology to this area of forensic analysis. In this method, 250nm was chosen for detection wavelength for the analytes rather than 220nm. It is because 220nm would have increased the sensitivity but not the selectivity. In this case, we emphasized the selectivity.
Gas chromatography coupled to mass spectrometry(GC-MS) employed in selected ion monitoring (SIM) is routinely used in forensic toxicological analysis. The application of this technique to the procedure shows that much lower levels of detection can be achieved by using this method of extraction.
The linear range of this method of extraction was found to be linear from 0 to 100ngmL-1. To measure the accuracy and precision of the method spiked samples were analyzed. In the analysis of spiked controls (40ngmL-1) blood samples were analyzed in duplicate. The values obtained by HPLC were: 34 (±5)ngmL-1 for the flunitrazepam and 48 (±5)ngmL-1 for the 7-aminoflunitrazepam.
The values obtained by GC-MS for spiked blood controls (40ngmL-1) were reported as: 37 (±4)ngmL-1 for the flunitrazepam and 45 (±4)ngmL-1 for the 7-aminoflunitrazepam. The recovery of flunitrazepam was 83% (±4%) and 87% (±4%) for 7-aminoflunitrazepam.
The limits of detection and quantification (LOD, LOQ) of the extraction method were determined using HPLC by undergoing a series of standards containing flunitrazepam and 7-aminoflunitrazepam with the internal standard (both unextracted and extracted from blood samples using solid phase columns) over a range of concentrations (5-100ngmL-1) to observe the lowest level at which the drug(s) could be detected. They were run on gas chromatography-mass spectrometry in selected ion monitoring mode with the addition of a decrease of five-fold in the lowest concentration level (5ngmL-1 of flunitrazepam and 7-aminoflunitrazepam). A detection level of 1ngmL-1 was achieved for both drugs (flunitrazepam and 7-aminoflunitrazepam) using gas chromatography-mass spectrometry in selective ion monitoring mode.
Analysis of a CAP proficiency test sample (urine) was found to be positive for 7-aminoflunitrazepam by Enzyme-linked immunosorbent assay (ELISA). The data supplied by the College of American Pathologists (CAP) indicated that the sample contained 63ngmL-1 of 7-aminoflunitrazepam (FTC-A Forensic Toxicology (Criminalistics), 2002). Analysis of the sample by this procedure using HPLC in 2004 revealed a level of 51 (±5)ngmL-1 of 7-aminoflunitrazepam. No flunitrazepam was detected. This showed that the method is valid for both internal and external controls.
It has been noted by earlier workers (Robertson and Drummer, 1998) that nitrobenzodiazepines, such as flunitrazepam are not only difficult to analyze cause of the low concentration of the drug that present in blood/urine samples (especially in DFSA cases which are discovered late after certain event) but also due the parent drug can break down to form the metabolite even after the sample has been drawn from the body and stored appropriately. In this situation, the concentrations of both flunitrazepam and the 7-aminoflunitrazepam are reduced even further.
Case study 2:
Analysis of explosives using high performance liquid chromatography with UV absorbance and photo-assisted electrochemical detection.
Explosives are the chemical compounds or mixtures that will, on application of an external stimulus such as heat, shock, friction or ignition, undergo rapid chemical decomposition. The chemical reaction causes sudden releases of large amount of energy due to liberation of gas and temperature. The pressure thus release out equally in all directions.
Although there are mechanical and nuclear explosives, chemical explosives are the most commonly used. A mechanical explosive is one in which a physical reaction is produced, like that caused by overloading a container with compressed air. While nuclear explosives, which produce a sustained nuclear reaction, are by far the most powerful, but their use is restricted due to the over powerful explosion. They normally used in certain industrial operation in oversea.
The main chemical explosives include black powder, dynamite, nitroglycerine and trinitrotoluene (TNT). A chemical explosive can be gaseous, liquid or solid. Nitroglycerin and dynamite succeeded black powder as the chief explosives. An Italian chemist, Ascanio Sobrero discovered nitroglycerin in 1846. The Swedish scientist, Alfred Nobel explored dynamite in 1867, the original explosives being a mixture of 75 % nitroglycerin and 25 % ghur (a porous, absorbent material that made the product easier to control and safer to use). Later, ammonium nitrate was used in dynamite which is more safer to use and cheaper to produce.
In order to analyse content of different explosive, several detection have been coupled to HPLC for the determination of explosives, including UV absorbance, refractive index(RI), mass spectrometry (MS) and dc amperometry (Yinon and Zitrin, 1993; Yinon 1990; Yinon, 1999; Krstulovic and Brown, P.R., 1982; Martens and Frankenberger Jr, 1900, Krull et al., 1983; Hilmi et al., 1999). However, RI and UV detectors are lack of sensitivity because of the presence of interfering compounds in environmental.
Ignoring its inherent flows, HPLC-UV is accepted and commonly used by the U.S. Environmental Protection Agency (EPA) for the determination of explosives in ground water and soil (EPA Method 8330). A ground water sample size of 1liter must be subjected to solvent extraction and be salted out and evaporated down to 5ml prior to analysis. Then samples be run on a C18 bonded-phase column and, subsequently, on a cyano bonded-phase column for confirmation of analyte identification. Reductive amperometry has been applied to the analysis of explosives to gain the selectivity. But it is suffer from lack of sensitivy and excessive noise, which is attributable to the reduction of dissolved oxygen present in the mobile phase and sample.
Post-column photochemistry in HPLC as a general derivatization approach for improved UV, fluorescence, and electrochemical detection has been used for a variety of analytes (Krull and LaCourse, 1986). Photochemical derivatization provides the advantages of a reagent-free system, including the elimination of mobile phase restrictions, pulsations due post-column delivery, inadequate mixing of post-column reagents, and matrix effects due to chemical reagents  I.S. Krull and W.R. LaCourse, Reaction Detection in Liquid Chromatography, Marcel Dekker, New York (1986) p. 326.(Krull and LaCourse, 1986). Photochemical reactors have variety of bulbs at different wavelengths and knitted reactor coils of various lengths and diameters. HPLC-photo-assisted electrochemical detection (HPLC-PAED), formerly referred to in the literature as HPLC-h?-EC, involves first the separation of the analytes of interest, followed by the photolytic generation of a new species that can then be detected electrochemically (Krull and LaCourse, 1986). For nitro compounds, it has been reported that photolysis results in bond cleavage to generate inorganic nitrite (NO2-) which is then oxidized at a glassy carbon electrode (no other species have been identified at this time) (Krull and LaCourse, 1986; Krull et al., 1983; Bratin et al., 1984; Krull et al., 1984; Krull et al., 1984; Krull et al., 1989; Selavka et al.,1986). Photo-assisted electrochemical detection (PAED), the detection scheme investigated here, operates in the oxidative mode and at applied potentials where dissolved oxygen is not a problem. Hence, PAED exhibits higher sensitivity than reductive amperometry.
The combination of HPLC-PAED known for its inherent sensitivity and selectivity for organic nitro compounds (Krstulovic and Brown, 1982; LaCourse, 1997), with on-line solid-phase extraction (SPE) for environmental samples in a compatible platform (Marple and LaCourse, 2003). The function of SPE is fractionate and concentrate the analytes of interest based on simple chromatographic principles. SPE eliminates sample handling and reduces dramatically (in combination with the increased sensitivity of PAED) the amount of sample required for analysis from 1L (required by Method 8330) to 2mL. It is this technique that makes the platform unique and compatible with the on-site analysis of explosives.
Reagents and solutions.
Purified water were used to made all solutions. Water was purified by using a reverse osmosis system coupled to multi-tank/ultraviolet ultrafiltration stations. All solvents are HPLC grade. All solvents were filtered with a Fisher vacuum filtration apparatus utilizing a 0.2µm PTFE membrane filter.
The standard solutions were purchased as mixtures denoted as either calibration mix 1 or calibration mix 2 at concentrations of 1000µg/mL each in 1mL acetonitrile. Table 1 lists the name, peak number, abbreviation, EPA classification (mix A or B), and Restek classification (1 or 2) of all the explosives of EPA Method 8330. Standard solutions were stored in a refrigerator at 4°C. All stock solutions were prepared daily. Ground water was obtained from Columbia Technologies (Baltimore, MD) and stored at 4°C until use.
The glassy carbon electrode was cleaned daily prior to use. The electrochemical cell is disassembled, and the working electrode is detached for polishing. A small amount of electrode polishing compound was poured onto a POLYPAD Gemstone Polishing Pad and placed the pad on a flat surface. The electrode was rubbed in a figure eight motion in the polishing compound for approximately 30s. It is important to hold the electrode flat against the pad to avoid rounding of the block which would result in cell leakage. All polishing compound were rinsed off from the working electrode with copious amounts of deionized water. Next, methanol was rinsed to remove oils deposited from the polishing compound. Follow up with a final rinsed of deionized water. All traces of polishing compound been removed because trace particulates on the electrode surface will alter electrode response. The cell was resembled and place it back into the LC-30 oven.
Nitro explosives are known toxins and carcinogens, so they should be handled with gloves in a fume hood. Skin, eye contact and ingestion must be avoided.
Result and discussion
The main compounds used in this experiment contributed from 14 nitro explosives. They are emphasis on RDX, TNT, and Tetryl which are commonly found at contaminated sites. EPA Methods 8330 utilizes UV detection at 254nm exploiting the inherent optical activity of each aromatic explosive. However, HMX and RDX both of them do not respond as well to this method because only compounds contain at least one nitro (-NO2) will show good reading by PAED.
Optimization for direct injection method
To optimize the PAED detection potential, the technique of hydrodynamic voltammetry (HDV) at a constant flow rate of 1.0mL/min was performed at the buffer pH values of 3.5,4.5 and 5.5 over a range of 0.2-1.0V. The resulting data was plotted as S/N (analytical signal-to-noise ratio) versus applied potential, as shown in Fig. 2A for HMX (-), RDX (cdots, three dots, centered), Tetryl (—), and TNT (··-··). From 0.7V the signal-to-noise start increasing slowly. The analytes show a maximum signal-to-noise ratio at 1.0V.. Beyond 1.0V, however, the analyte response begins to decrease while the noise increases, thus decreasing the signal-to-noise ratio. Based on the result, HMX and RDX show the higher signal-to-noise raiot. It is due to the fact the N-N bonds in nitramines are much weaker than the C-N bonds of nitro aromatics), and the photolytic cleavage of N-N bonds is much more efficient. As result more nitrite was formed from nitramines than from nitro aromatics. Therefore higher S/N ratio detected for HMX and RDX. Varying the pH showed little effect, so a pH of 4.5 and applied potential of 1.0V were chosen as optimum values for PAED.
Full-size image (12K)
Here, the potential used was 1.0V and pH=4.5, and the flow rate was varied between 0.4 and 1.4mL/min based on Fig. 2B. At flow rates below ca. 0.8mL/min, chemical degradation of electroactive species occurs due to the residence time in the photoreactor is too long (Krull, I.S et al., 1983). The analytes show a maximum signal-to-noise ratio at 1.0mL/min, and all faster flow rates do not allow enough time for the generation of the maximum amount of photoproducts. All nitro compounds responded similarly to those shown in Fig. 2B, and an optimum flow rate of 1.0mL/min was chosen.
Fig. 2.(A) Hydrodynamic voltammograms for HMX (-), RDX (cdots, three dots, centered), Tetryl (—), and TNT (··-··) at pH=4.5. Mobile phase: 50% methanol in 20mM acetate buffer, pH=4.5; flow rate: 1.0mL/min; guard column: Phenomenex SecurityGuard with 4mm-3.0mm C8 cartridge; column: C18, 5µm, 4.6mm-250mm; column oven temperature: 30°C; electrode: 1.0mm glassy carbon; reference electrode: Ag/AgCl. (B) Plot of electrochemical S/N vs. flow rate for HMX (-), RDX (cdots, three dots, centered), Tetryl (—), and TNT (··-··). Mobile phase: 50% methanol in 20mM acetate buffer, pH=4.5; guard column: Phenomenex SecurityGuard with 4mm-3.0mm C8 cartridge; column: C18, 5µm, 4.6mm-250mm; column oven temperature: 30°C; electrode: 1.0mm glassy carbon; reference electrode: Ag/AgCl; applied potential: 1.0V vs. Ag/AgCl.
Optimization of on-line SPE
In order to perform on-line SPE, sample is loaded into the 2mL injection loop, and the prep injector is electronically actuated, which allows the SPE solvent to flow through the loop and carry the sample to the SPE column. The prep injector is returned to the load position after an optimized “wash” time that concentrates the analyte and rinses off potential contaminants, while the 6 port valve is turned to the inject position simultaneously. This procedure allows the HPLC mobile phase to backflush through the precolumn and elutes the analytes of interest onto the analytical column. This valve remains open throughout the entire chromatographic run. At the end of the run, a 5min wash with 80% methanol cleans the SPE column, which is followed by a 5min equilibration with “wash” solution prior to the next injection.
A wash solvent of 7.5% methanol in a solution of 20mM sodium acetate trihydrate (pH=4.5) and 0.5M sodium chloride was determined to give the greatest sample cleanup. As in the “salting out” extraction process of EPA Method 8330, sodium chloride was added to increase the retention of the explosives from the matrix into the C18 phase of the SPE column. The methanol was needed to wet the C18 phase, and the sodium acetate buffer was added to matrix match the wash solvent and the HPLC mobile phase.
Direct injection of explosives
The UV and PAED chromatograms under optimized separation and detection conditions are shown in Fig. 3 (A: UV; B: PAED). The use of two detectors show three distinct modes of selectivity. First, inherent to all chromatographic methods, is the selectivity afforded by the chromatography and the comparison of retention times of standards versus analytes in a sample. This is due to the nitro compounds cannot be oxidized directly, no detectable signal for the compounds of interest when the lamp in the photochemical reactor is turned off. Next, the second mode of selectivity is that the analyte must be photoreactive and produce oxidizable products to be detected. Finally, the use of two detectors allows for the determination of response ratios for standards and those for analytes in a sample.
Fig. 3.Optimized separation of explosives: (A) UV at 254nm chromatogram; (B) PAED chromatogram. Mobile phase: 50% methanol in 20mM acetate buffer, pH=4.5; flow rate: 1.0mL/min; guard column: Phenomenex SecurityGuard with 4mm-3.0mm C8 cartridge; column: C18, 5µm, 4.6mm-250mm; column oven temperature: 30°C; electrode: 1.0mm glassy carbon; reference electrode: Ag/AgCl; applied potential: 1.0V vs. Ag/AgCl.
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Upon examination of the two chromatograms (Fig. 3A and B), the expected increased sensitivity achieved by PAED for the nitramines (HMX and RDX) and other compounds such as Tetryl and TNT are easily seen. The figures of merit for the model compounds are presented in Table 3A. These were determined using the direct injection method with the 100µL injection loop, and all solutions were made in deionized water. When comparing limits of detection, PAED (LODs 0.007-3µg/L) is more sensitive than UV detection (LODs 0.9-5µg/L) for almost all of the model compounds. For some of the more common explosives, there is an approximate 1000- and 100-fold increase in sensitivity for HMX and RDX, respectively, and an approximate 100- and 10-fold increase in sensitivity for Tetryl and TNT, respectively, over UV detection. Furthermore, the PAED limits of detection are much lower here than those previously described for h?-EC . (Yinon, 1999; Krull et al., 1983; Krull and LaCourse,1986; Krull et al., 1983; Bratin et al., 1984; Krull et al., 1984; ,  I.S. Krull, C. Selavka, X-D. Ding, K. Bratin and G.A. Forcier, Curr. Sep. (1984) (5), p. 57.Krull et al., 1989; Selavka et al.,1986)
The slope of the line (m) and the y-intercept (b), were included to show the calibration sensitivity of the detection methods and that there is no system bias in the method, respectively. And also, R2 values determined by linear regression analysis show that UV and PAED are of comparable linearity over the concentration range tested, which is at least four orders of magnitude in both cases. The percent relative standard deviation (%R.S.D.), determined by seven injections at the approximate limit of quantitation for each explosive, ranged from 0.80 to 3.41% for UV detection and 0.46 to 6.70% for PAED, all under 15% as required by the Resource Conservation and Recovery Act (RCRA) were described in Section 3.5.
On-line SPE analyses
A large background was present on the electrochemical detector that completely overwhelmed any signal from the explosives present when a real ground water sample was injected on the existing system, as shown in Fig. 4A. This is due to the electroactive species present in the groundwater (e.g., urea, salt etc.). PAED limits the analyzation of these species, therefore on-line SPE was chosen for reasons stated earlier. When those electroactive species eliminated, the high background was virtually eliminated, as seen in Fig. 4B.
Table 3B displays the figures of merit for the nitro explosives in deionized water using the SPE system. Preconcentrated limits of detection ranged from 0.0007 to 0.4µg/L for PAED and from 0.04 to 0.4µg/L for UV detection. It should be noted that on-line SPE could be used with Method 8330 alone to increase its sensitivity without incorporating PAED, but PAED should be used for Method 8330 enhancement. This concentration factor will turn the need of 1L of ground water sample to 2mL water sample. Linearity is retained when using SPE, with an average R2 value of 0.99962 (PAED and UV). The %R.S.D.s ranged from 1.17 to 5.38%, comparable to those by direct injection and still within the Resource Conservation and Recovery Act (RCRA) guidelines.
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