Selective spectrophotometric determination of trinitrotoluene, trinitrophenol, dinitrophenol and mononitrophenol

Analytica Chimica Acta 505 (2004) 83–93

Selective spectrophotometric determination of trinitrotoluene, trinitrophenol, dinitrophenol and mononitrophenol Aysem Üzer, Erol Erça˘g, Resat Apak∗ Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar, 34850 Istanbul, Turkey Received 25 October 2002; received in revised form 9 May 2003; accepted 22 May 2003

Abstract Contaminated land and groundwater remediation in military waste dumping sites often necessitates the use of simple, cost-effective and rapid tests for detecting trinitrotoluene (TNT) and trinitrophenol (picric acid; PA) residues in the field along with their dinitro-analogues. Using PA as the model compound, a simple and field-adaptable (on-site) colorimetric method was developed for quantifying PA in the presence of dinitrophenol (DNP) and mononitrophenol (NP). Most commercialized methods for TNT assay—with the exception of CRREL method—use proprietary chemicals, and the color stability and intensity are highly dependent on the composition of the organic solution comprised of acetone or methanol. The developed colorimetric method here is based on the extraction of the Meisenheimer anion formed from the reaction of PA and aqueous NaOH into isobutyl methyl ketone (IBMK) with a cationic surfactant such as cetylpyridinium bromide (CPB). The orange-red color that developed in the organic phase was persistent for at least 30 min. TNT formed a similar extractable red complex under these conditions. If present, 2,4-dinitrophenol (DNP) and 4-nitrophenol (NP) could be detected by the same method at 17and 167-fold concentrations of the LOD of PA, i.e. 1.5 ppm. DNP alone could be quantified by another charge-transfer (CT) agent, imidazole, as a yellow product at 400 nm in 98% EtOH solution. Under the same conditions, the intramolecular CT-band due to PA was essentially not intensified upon addition of the imidazole ligand, enabling the estimation of the DNP concentration from absorbance difference of solutions with and without imidazole, due to the intermolecular CT absorption of the latter. NP alone could be detected with a diphenylamine solution in H2 SO4 to produce a blue color. © 2003 Elsevier B.V. All rights reserved. Keywords: TNT; Trinitrophenol; Picric acid; Dinitrophenol; Determination; Polynitroaromatic explosives; Spectrophotometry; On-site colorimetry

1. Introduction The explosive polynitroaromatic compounds such as trinitrotoluene (TNT), trinitrocellulose and trinitrophenol (picric acid) can be analyzed and differentiated from lower nitro-analogues usually by sophisticated instrumental techniques such as RP-HPLC (e.g. method 8330 using an LC-18 (octadecylsilane) column with UV detection at 254 nm [1]) and size-exclusion chromatography coupled with FTIR or MS. The requirements for quick decision making in criminology laboratories or reclaimed military sites (contaminated with TNT residues or its degradation products) often impose the use of simple, cheap and selective field techniques such as colorimetry and spectrophotometry. On the

∗

Corresponding author. E-mail address: [email protected] (R. Apak).

0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0003-2670(03)00674-3

other hand, spectrophotometric methods specific for reactive nitro-groups do not differentiate the higher nitro-compounds from the lower analogues, which is an essential criterium for explosibility. Because of the extremely heterogeneous distribution of explosives in contaminated soils of abandoned and to be remediated military sites, on-site analytical methods are a valuable, cost-effective tool to assess the nature and extent of contamination. Because costs per sample are lower, more samples can be analyzed and the availability of near-real time results permits redesign of the sampling scheme while in the field. On-site screening also facilitates more effective use of off-site laboratories using more robust analytical methods [2]. The first on-site method of detecting explosives residues in water was reported by Heller et al. in 1982 [3] and later improved by Erickson et al. in 1984 [4] who extended its application to include soils. This method was specifically aimed at the detection of TNT and utilized

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a detection tube that had two sections. The first section contained a basic oxide (CaO) that converted TNT to its Meisenheimer anion. This colored species migrated to the second section of the tube, where it was retained on a quaternary ammonium-type anion exchange resin. Water samples were pumped through this tube, and TNT was detected visually by the development of a reddish stain on second section of the tube, the concentration of TNT being proportional to the length of stain produced. The colored TNT anion was sensitive to daylight and even common anions at high concentrations such as Cl− and NO3 − produced purplish stains, preventing the formation of the TNT anion [3]. The ability of the method to precisely and accurately quantify TNT concentrations in water and soil was evaluated as poor by CRREL [5]. Another on-site method developed by Stevanovic and Mitrovic for TNT (and RDX) was based on passing water through a porous disk coated with a thin film of silica gel, enabling adsorption of the analyte on the surface [6]. The disk was dried and sprayed with a color forming solution of o-toluidine for TNT, which is rather an unspecific reagent capable of forming charge-transfer complexes with many nitroaromatics, and measurement was made by reflectometry. The fiber-optic-based approach of Zhang et al. utilized the reaction of TNT with an amine-loaded PVC membrane to form a colored product [7]. Finally colorimetric-based methods of TNT and RDX in water and soil were developed at CRREL by Jenkins and coworkers based on acetone extraction of soil, reacting with a base (NaOH) and reducing agent (Na2 SO3 ) to produce the highly colored Janowsky anion absorbing at 540 nm [8–11]. The reaction equations for the formation of the Meisenheimer anion from TNT CH3

CH3 NO2

O 2N

NO2

O 2N OH-

+

-

OH H

NO2

NO 2

or the Janowsky anion in the presence moisture-free acetone and tetrabutylammonium hydroxide CH3

CH3 NO 2

O 2N

certain chemicals (such as the reducing agent Na2 SO3 that is claimed to take part in Janowsky anion formation from TNT) in moisture-free acetone, colorimetric procedures based on Meisenheimer formation should be dependent on less parameters than those based on Janowsky anion formation to produce a lasting color that can be measured within a reasonable period of time. When polynitroaromatics are reacted with alkali hydroxides, a ‘Meisenheimer anion’ forms from the parent aromatic compound of color changing from violet to red [13]. Picric acid (PA or trinitrophenol) or TNT may react with NaOH in aqueous–organic mixture solutions to form the orange-red colored Meisenheimer anion, and this reaction forms the basis of the currently available CRREL spectrophotometric method for TNT assay, the commercialized EnSysRISc version of which is sold in the market as reagent kits without accessible contents [9,14]. Unfortunately, Jenkins’ colorimetric method (also known as the CRREL method) does not involve a stable color in acetone–water solution unless the solution is filtered, and the operator has to measure the color intensity within 3–8 min in unfiltered solution [8,9]. The color formation is highly dependent on the acetone (or methanol) content of the medium, and the relative instability of the product is open to high systematic errors that may arise from operator faults. Moreover, the patented nature of the reagent kits may cause high costs of analytical services (such as 15 USD per sample) [2] in monitoring TNT contaminated land and water remediation operations. To overcome the color stability problem, the two-column apparatus filled with a basic oxide and quaternary ammonium anion-exchanger resin designed to stabilize and measure the Meisenheimer anion of TNT in the field gave results highly dependent on column length and NaOH concentration [15]. On the other hand, the CRREL field method for quantifying ammonium picrate and picric acid in soil and water [16] is based on the retention of picrate ions from water or acetone extract of soil on an anion-exchanger or alumina column by solid phase extraction (SPE), eluting the retained picrate as picric acid, and converting back to a colored picrate solution in a 1:3 water–acetone mixture, followed by absorbance measurement at 400 nm. Since other substances present in soil producing yellow extracts (such as soil humic acids)

-

+

H 2C

O C

NO 2 O

O 2N CH3

-

CH2

C

CH3

H NO2

NO 2

were hypothesized as above in review literature [12] to form the basis of CRREL and USACE methods, and of some commercialized colorimetric procedures such as EnSysRISc and ENVIROL, the latter two using proprietary chemicals for color development [2]. Since soil extracts naturally contain some humidity, and it is extremely difficult to dissolve

interfere with the CRREL picric acid assay, a background correction is necessary [16]. Thus the aim of this work is to develop a mutually beneficial color reaction for both TNT and PA showing similar chemical behavior, to stabilize the Meisenheimer anion formed from PA, and to develop a stable color reaction suitable for field use in all conventional

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laboratories dealing with polynitro-explosives and their possible degradation products, e.g. the bacterial degradation of TNT in the field gives rise to dinitro-aromatics. The developed colorimetric method is based on the stabilization of the colored anion formed from alkali-reacted TNT or PA by extraction with a cationic tenside (CPB) into IBMK, and measurement of the absorbance in the organic phase. Dinitro- and mononitrophenol are assayed by making use of different charge-transfer complexes.

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extract of a solution containing all reagents but TNT). The calibration line was drawn for 4–40 ppm initial solutions (or 2.67–26.67 ppm final solution in IBMK) of TNT. Since the procedure is water-tolerant (i.e. many reagents are aqueous solutions), the initial TNT solution need not necessarily be in acetone (i.e. a special requirement of the Jenkins’ method [8,9]), and the developed method is applicable to TNT analysis in aqueous solution. 2.4. Application of the Jenkins’ method [8,9] and developed procedure to TNT assay in soil

2. Experimental 2.1. Materials and methods All reagents were supplied from Merck, Darmstadt (with the exception of humic acid, Fluka), and were of analytical reagent grade. A TNT sample was kindly provided by Istanbul Police Headquarters, Criminology Laboratory. The PA-added soil (white clay soil) was sampled from the courtyard of Istanbul University, Avcilar Campus. The TNT-added clay soil composed of (by mass percentage) 25.76% sand, 64.35% clay and 9.89% dust, was provided by the Faculty of Forestry, Istanbul University Sariyer Campus. The spectrophotometric measurements, either in homogeneous solution or as extractive-photometry in the organic (IBMK) phase, were made with a Cary 1E UV-Vis spectrophotometer, working either in the main or derivative spectral mode. 2.2. Application of the Jenkins’ method [8,9] to standard TNT solutions Standard TNT solutions of varying concentration (between 0.1 and 15 ppm) were prepared in acetone containing 4% H2 O (i.e. the minimum amount stated in the method for reagent solubility). To 25 ml of the standard solution were added a medium size pellet of KOH and 0.2 g anhydrous Na2 SO3 , and agitated for color development in ultrasonic bath for 5 min. The undissolved reagents were filtered off through a double-fold blue band (Whatman) quantitative filter paper, and the absorbance of the homogeneous solution was read at 540 nm against a reagent blank. The calibration line was drawn for standard solutions.

The finely ground standard clay soil (Faculty of Forestry, Istanbul University) was dried in an oven at 110 ◦ C, and the weakly bound water (humidity) content was found as 4.16%. To 20 g of this dry soil were added 10 ml of TNT solution (in acetone) of varying concentration between 10 and 100 ppm, well mixed and homogenized, and let to stand at room temperature for drying. The soil was leached with 100 ml acetone (containing 4%, v/v H2 O) for 10 min in a stoppered flask so that the TNT content of soil was extracted with an efficiency of ≥90% [8,9]. The leachate was filtered through a Nr. 2 Gooch crucible, and to a 25 ml-aliquot of the extract were added a medium size KOH pellet and 0.2 g Na2 SO3 , agitated for 5 min in an ultrasonic bath, filtered through a double-fold blue band filter paper, and the absorbance of the filtrate was read at 540 nm against a reagent blank. (The reagent blank of the uncontaminated soil leachate showed an absorbance of ∼ =0.01 against acetone.) From the acetone leachate of soil, a 5 ml-aliquot was drawn, 0.5 ml of 5% NaOH and 4 ml of 7.5 × 10−3 M CPB were added, the mixture extracted with 7.5 ml IBMK, the organic extract filtered through a double-fold blue band filter paper, and the absorbance of the filtrate was read at 500 nm against a reagent blank (the reagent blank of the uncontaminated soil extract showed an absorbance of ∼ =0.01 against IBMK). 2.5. Investigation of the water tolerance of TNT assay methods Varying amounts of water were added to 10 ppm acetone solutions of TNT such that the water content varied between 6 and 25% (v/v), and both the Jenkins’ and developed methods were applied.

2.3. Application of the developed method to standard TNT solutions

2.6. Studying the role of Na2 SO3 in color development

To 5 ml of the standard TNT solution in 1:1 acetone–water were added 0.5 ml of 5% aqueous NaOH and 4 ml of 7.5 × 10−3 M aqueous cetylpyridinium bromide (CPB) solution, and extracted with 7.5 ml isobutyl methyl ketone (IBMK). The extract was filtered off through a double-fold blue band (Whatman) quantitative filter paper for removal of any water droplets, and the absorbance of the red-colored IBMK extract was read at 500 nm against a reagent blank (i.e. organic

The Jenkins’ method was applied to TNT solutions with and without sodium sulphite as a reagent of the color development medium. The definite basicity of 0.2 g anhydrous Na2 SO3 in 25 ml solution as practiced in the Jenkins’ method [8,9] was theoretically calculated assuming that the solution was aqueous for the sake of simplicity, and similar basic mixtures in conjunction with KOH were prepared using either Na2 HPO4 or NaHCO3 mixed with one-tenth of Na2 CO3

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in order to investigate the true origin of the color intensification effect reported [8,9], i.e. as regards to sulphite’s possible reducing ability or its enhanced basicity. 2.7. Investigation of Cu(II) and humic acid interferences Copper(II) and humic acids in soil were reported to interfere with the CRREL method of TNT and DNT assay [8,9]. A540 of 10 ppm TNT solutions in the Jenkins’ method was measured as a function of Cu(II) content in the presence of 30-fold Cu, followed by A500 measurements in the developed method. A suitable volume of 0.2% (w/v) humic acid in aqueous alkaline solution was added to a 10 ppm TNT solution in the Jenkins’ method without exceeding the critical limit of H2 O ratio, such that the absorbance (at 540 nm) increased by ≥50%. The same amount of humic acid was tested for its possible interference with the developed extractive-photometric method. 2.8. Application of the CRREL method of PA assay [16] to standard PA solutions The standard CRREL method for PA assay in soil is described as acetone leaching of soil, filtering, diluting with H2 O thereby converting PA to picrate ions, passing the solution through an anion-exchanger resin or alumina column to retain picrate, removing interferences with methanol washing, eluting picrate as picric acid with H2 SO4 -acidified acetone, recording the initial absorbance at 400 nm (Ai ) for background correction (from soil humates and other possible interferents), converting the colorless PA solution to a yellow-colored picrate solution by diluting with unacidified acetone and water, remeasuring the absorbance at 400 nm (Af ), and finally correlating the net (corrected) absorbance (Af − Ai ) with the amount of PA in soil [16]. Assuming 100% adsorption and elution of PA onto-and-from the ion-exchanger column (a target which may hardly be achieved in real analytical separations), this method was applied to standard PA solutions in 1:3 (v/v) water–acetone mixtures. 2.9. Application of the developed method to standard PA solutions and PA-added soil Exactly the same procedure was followed as described for TNT solutions in 1:1 acetone–water. The only difference is that the absorbance at 490 nm of the orange-colored IBMK extract was measured against a reagent blank. To observe possible interference effects, 2,4-dinitrophenol (DNP) and 4-nitrophenol (NP) were subjected to the same procedure as that of PA. For PA analysis in soil, 2 g of a completely dried soil sample (white clay soil, from the courtyard of Istanbul University at Avcilar Campus) was treated with 1.0 mg PA (i.e. 10 ml of 100 ppm PA solution) in acetone–water mixture such that PA was thoroughly impregnated to soil. The PA-sorbed soil

was dried at 80 ◦ C in an oven, and mixed well for homogenization. A 0.5 g sample was taken from this homogeneous soil, and extracted with 10 ml acetone for 15 min; 10 ml H2 O was added, and agitation was continued for 10 more min. The suspension was filtered through a blue-band filter paper, and 5.0 ml of the filtrate was taken for analysis by the developed procedure. For testing the possible interference of soil humates, a suitable volume of 0.2% (w/v) aqueous alkaline solution of humic acid was added to 1:3 water–acetone solution of 25 ppm PA such that the yellow solution turned orange-brown (i.e. more than 50% increase in A400 ). The same amount of humic acid solution was added to 25 ppm PA solutions in the developed extractive-photometric method of PA assay, and A490 of the IBMK extract was measured. 2.10. Dinitrophenol assay with the developed (main and derivative) spectrophotometric procedures For DNP assay in a separate procedure, 5 ml of DNP solution in 98% C2 H5 OH was reacted with 5 ml of a 1000 ppm ethanolic (98% C2 H5 OH) solution of imidazole, and the ordinary (at 400 nm) as well as second derivative (2 D) absorbance (at 426 nm) values of the yellow colored charge-transfer complex (CTC) were recorded. A wavelength difference (λ) of 10 nm was taken for second derivative spectra. Since the yellow color of DNP was intensified upon the addition of imidazole to the ethanolic solution whereas a similar color of PA was almost maintained, the difference in absorbance (A) of alcoholic DNP solutions with and without the electron-donating imidazole ligand could be used as a parameter to be correlated to DNP concentrations, and hence to estimate the DNP content of (PA + DNP) mixtures without working in the derivative mode (this may also be more applicable to field use than derivative spectrophotometry). To find the suitable imidazole concentration for observing this correlation, 25 ppm DNP was treated in equal volumes with 50–1000 ppm imidazole solutions in absolute alcohol (EtOH), and the absorbances (A) recorded at 400 nm were compared with those of respective DNP solutions not containing imidazole, A being defined as the difference. The difference in absorbance (A) at 400 nm of 1000 and 0 ppm imidazole-containing solutions of DNP were recorded as a function of analyte concentration for DNP assay without derivative spectrophotometry. 2.11. Qualitative detection of mononitrophenol Mononitrophenol (NP) was qualitatively determined with a sulfuric acid solution of diphenylamine (DPA). The reagent was prepared by dissolving 1 g DPA in 100 ml of concentrated H2 SO4 . The period for color development of reagent-added NP solution was 5 min. The development of a blue color indicated the presence of NP.

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3. Results and discussion The methods developed for TNT or PA assay in soil or aqueous solutions are based on the stabilization of the orange-red colored Meisenheimer anions of TNT or PA by solvent extraction of the ion-associates formed in strongly alkaline medium with the cationic tenside CPB into IBMK, with subsequent absorbance measurement in the organic phase (at 500 nm for TNT, and at 490 nm for PA). This is an improvement over literature methods which measure TNT in soil extract based on Meisenheimer or Janowsky anion formation [8,9,14] highly dependent on aqueous phase composition, or over the CRREL method which measures PA in soil extract without making use of a Meisenheimer anion [16]. The developed field-adaptable extractive-photometric method, mutually applicable to TNT or PA assay in soil and water, is also a good alternative to the more costly and sophisticated instrumental methods which are not readily available to conventional laboratories. Moreover, PA assay by the developed method could effectively distinguish it from lower nitrophenols (such as DNP and NP). 3.1. TNT determination The Jenkins’ method [8,9] applied to standard acetone solutions (N = 5 measurements) of TNT (in the range: 0.1–15 ␮g ml−1 ) gave the calibration line: A540 = 5.52 × 10−2 CTNT + 1.47 × 10−2

(r = 0.995)

where CTNT is the ␮g ml−1 concentration of TNT in solution. The LOD of the method was 0.12 ␮g ml−1 , with an effective molar absorptivity of ε = 1.25×104 l mol−1 cm−1 (in acetone containing 4% water). When the developed method was applied to TNT standard solutions in acetone–water in the range 4–40 ␮g ml−1 (or between 2.67 and 26.67 ␮g ml−1 in the IBMK extract), the calibration line for initial concentrations was: A500 = 2.32 × 10−2 CTNT + 1.02 × 10−2

(r = 0.9995)

where CTNT is the ␮g ml−1 concentration of TNT in solution. If this calibration line was drawn in terms of TNT extracted to the IBMK phase, the equation was: A500 = 3.48 × 10−2 CTNT + 1.01 × 10−2

(r = 0.9995)

where the molar absorptivity for TNT (in IBMK extract, or final solution) was ε = 7.90 × 103 l mol−1 cm−1 , with a LOD of 0.13 ␮g ml−1 . Although the Jenkins’ method has a higher molar absorptivity at a higher (red-shifted) wavelength, the precision is significantly lower probably due to the different alkalinity levels achieved with respect to the KOH pellet size (as aqueous solutions of KOH may not be used in the Jenkins’ method). The color intensity developed in the homogeneous solution is highly dependent on the water content of solution, and the molar absorptivity decreases drastically with increasing water ratio.

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Table 1 Dependence of the color intensity of the Jenkins’ method on the water content of the acetone solution of 10 ppm TNT Water content (vol.%)

A540

4 6 15 17.4 20 25

0.580 0.520 0.372 0.369 0.123 0.0962

When the Jenkins’ (CRREL) method was applied to TNT-containing clay soil (in the range: 5–50 ppm TNT in soil, corresponding to 1–10 ␮g ml−1 TNT in final solution), the calibration line (for N = 5 measurements) was: A540 = 1.36 × 10−2 CTNT − 0.029 (CTNT : ppm concentration in soil; r = 0.981) with a low reproducibility and a high R.S.D. Naturally this worked for filtered solutions with water contents of the acetone extracts optimized at 4%; in other cases, both the absorptivity and the precision were lower. The developed method, applied to the same soil standards, yielded: A500 = 5.35 × 10−3 CTNT + 3.25 × 10−2 (CTNT : ppm concentration in soil; r = 0.999) with a higher reproducibility. It should be noted that the molar absorptivities of both methods for soil leachates were about 1.15–1.2 times higher than those found for standard solutions. The LOD values for TNT in soil standard samples were 1 and 2.7 ppm (␮g g−1 ) for the Jenkins’ and the developed method, respectively. Varying amounts of water were added to 10 ppm (acetone) solutions of TNT such that the water content (by vol.) varied between 6 and 25% (A 10 ppm solution of TNT in 4% H2 O-containing acetone medium of maximum absorption [8,9] typically yielded an absorbance at 540 nm of 0.58±0.02.) As seen from Table 1, the absorbance at 540 nm decreased drastically (in a non-linear fashion) with increasing water content. The spectra of the above solutions are shown in Fig. 1, demonstrating that the Jenkins’ (CRREL) method has a low water tolerance, as the charge-transfer bands forming the basis of colorimetric detection are obscured by the presence of water. Increased water content in acetone media decreased both the color intensity and color stability (i.e. with respect to time, figure not shown) of the Jenkins’ method. This is why the method [8,9] is only applicable to soil leachates not exceeding a predetermined H2 O ratio, and not to water samples of TNT. On the other hand, in the developed method, a 10 ppm (initial concentration) TNT solution yielded an absorbance at 500 nm of 0.24 ± 0.02 when the water content of the medium was increased from 6 to 50%. Thus the recommended method is

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A. Üzer et al. / Analytica Chimica Acta 505 (2004) 83–93 0,9 6%

0,8

15%

Water content: (by vol.)

0,7

17.4% 20%

Absorbance

0,6

0,5

0,4

0,3

0,2

0,1

0 400

450

500

550

600

Wavelength (nm)

Fig. 1. The spectra of 10 ppm TNT solutions in acetone containing varying percentages of water, obtained by application of the CRREL method [8,9].

capable of being applied to extremely humid soils without a need to dry the sample, and to aqueous solutions. The effect of Na2 SO3 , an indispensable reagent of the Jenkins’ method, was investigated in detail. Three standard solutions of 10 ppm TNT, with and without Na2 SO3 , gave similar absorbances in the method, i.e. A540 = 0.58 ± 0.02. Thus, the possible role of sulfite as a reducing agent capable of binding to the aromatic ring (of the TNT–Meisenheimer anion) is questionable. The contribution of SO3 2− to the basicity of the presumably aqueous medium (i.e. KOH + Na2 SO3 ) may be calculated from the formula: pH = 21 (pKa2 + pKw + log CSO3 2− ) where pKa2 is the second acidity constant of H2 SO3 , pKw is the autoprotolysis constant of water, and CSO3 2− is the molar concentration of sulfite (present as 0.2 g in 25 ml solution). Since the calculated pH was about 9.1, similar pH conditions were generated by replacing Na2 SO3 as a reagent with either Na2 HPO4 (the pH of which is approximately equal to the arithmetic mean of the second and third acidity constants as pKa2 and pKa3 of H3 PO4 ), or with a suitable Na2 CO3 + NaHCO3 mixture (pH = pKa2 + log(CCO3 2− /CHCO3 − )), where pKa2 is the second acidity constant of H2 CO3 , and CSO3 2− and CHCO3 − are the molar concentrations of carbonate and bicarbonate, respectively. In each case, the absorbance was (within experimental error) around the value expected from a KOH + Na2 SO3 mixture as recommended by the Jenkins’ method. On the other hand, when sole NaHCO3 was used in place of Na2 SO3 ,

A540 significantly decreased, because the calculated pH due to only HCO3 − ion in a presumably aqueous solution is ca. 8.3 (i.e. less than that achievable with Na2 SO3 ). Therefore it was concluded that an additional reducing effect due to Na2 SO3 to cause a higher degree of charge transfer to possibly form a Janowsky anion was not observable under the employed experimental conditions, and the effect of sulfite possibly originated from its enhanced basicity to form a Meisenheimer anion when used in conjunction with KOH. The presence of Cu(II), reported as an interferent in DNT assay via binding to the –NO2 groups [9], yielded slightly fluctuating absorbances in the Jenkins’ method of TNT assay (e.g. up to 30-fold Cu in the analysis of 10 ppm TNT), and Cu(II) was reported as an interfering cation in the original method [9]. In the developed method, Cu(II) gave negligible absorbance deviation up to 30-fold amounts but obstructed the extraction of 10 ppm TNT (into IBMK) at 50-fold concentration limit. This probably arose from the reaction of Cu(II) with KOH, thereby consuming the alkalinity of the medium and preventing the formation of an extractable Meisenheimer anion of TNT. Soil humates, either free or metal-bound, may cause positive interference to the Jenkins’ method of TNT assay as long as they pass to the acetone leachate of soil containing a small amount of water. A suitable amount of 0.2% aqueous alkaline solution of humic acid causing ≥50% positive interference in the Jenkins’ method (i.e. A540 increased from 0.56 to 0.85) was added to 10 and 20 ppm TNT solutions

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in the developed extractive-photometric method, and the absorbances (A500 ) of the IBMK extracts were 0.24 and 0.47, respectively (i.e. the expected values in the absence of humic acid). Humates did not interfere with the developed extractive-photometric method, because these substances comprised of polymeric phenols and carboxylic acids are expected to be multivalently anionic at the high pH employed, and multivalent anions may not be extracted due to high hydration energies (as opposed to the preferential extraction of univalent TNT anion with the univalent cetylpyridinium cation). 3.2. Picric acid determination Picric acid determination by the CRREL method [16] depends on the ion-exchange separation of PA from the soil extract, and measurement of A400 of the yellow solution due to picrate ions in 1:3 water–acetone mixture. Charge-transfer due to the acidic dissociation of PA in acetone–water is found sufficient for measurement in the selected solvent medium without necessitating the presence of a strong base. However, the method is open to interferences of other substances in soil that show a similar ion-exchanger retention/elution behavior to that of PA, and at the same time produce yellow extracts. The CRREL method was applied to PA solutions in acetone–water of concentration range: 1–15 ppm (N = 5 measurements), and the equation of the calibration

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line was: A400 = 5.72 × 10−2 CPA − 1.88 × 10−2

(r = 0.9999)

where CPA is the ppm (␮g ml−1 ) concentration of PA. The developed extractive-photometric method was applied to PA solutions of initial concentrations in the range: 5–75 ppm (or 3.33–50 ppm in IBMK extract), and the equation of the calibration line (as a result of N = 6 measurements) was: A490 = 9.21 × 10−3 CPA + 6.16 × 10−3

(r = 0.9998)

where CPA is the ppm (␮g ml−1 ) concentration of the initial solution. The LOD for PA assay in solution was 0.4 ppm. If this calibration line was drawn in terms of PA extracted to the IBMK phase, the equation became: A490 = 1.38 × 10−2 CPA + 6.16 × 10−3 where CPA is the ppm (␮g ml−1 ) concentration of PA in final solution (IBMK extract), providing an effective molar absorptivity of ε = 3.16 × 103 l mol−1 cm−1 . While the CRREL method yielded a monotonous spectrum up to the UV range with no useful band in the visible region, the developed extractive-photometric method gave a distinctive shoulder at 490 nm utilized for quantitative measurement (Fig. 2). Such a characteristic spectrum, as used in the developed method, is much more beneficial for solving interference problems due to soil components producing

1 0,9

0,8

Absorbance

0,7 0,6

0,5

0,4 0,3

0,2 0,1

0 400

450

500

550

600

Wavelength (nm)

Developed method, 50 ppm PA in IBMK extract Developed method, 15 ppm PA in IBMK extract CRREL method, 10 ppm PA in 1:3 water-acetone mixture

Fig. 2. The spectra of PA solutions, both in 1:3 water–acetone mixture according to the CRREL method [16], and in IBMK extract of the ion-associate formed from PA–Meisenheimer anion and CP cation according to the developed method.

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yellow extracts (such as humic acid) which would otherwise require background corrections as stated in the original CRREL method [16]. A suitable volume of 0.2% aqueous alkaline solution of humic acid was added to a 1:3 water–acetone solution of 25 ppm PA such that the yellow solution turned orange-brown (i.e. more than 50% increase in A400 of the CRREL method). The same amount of humic acid solution was added to 25 ppm PA solution in the developed method, extracted with CPB/IBMK, and the absorbance of the extract at 490 nm was 0.24, without any deviation from that of a similar standard without humic acid. This showed that humic acids, being macromolecular phenolic carboxylic acids, were multivalently charged anions at the high pH employed for PA assay, and therefore were not extracted along with PA to cause any interference in the developed method. The LOD level achieved for PA is perfectly in accord with preliminary remediation goals of TNT/PA contaminated soils [15]. An orange-red color was not formed with DNP and NP under the same conditions. However, under extremely high concentrations, these nitro-compounds showed interference in that DNP and NP could be tolerated at 17- and 167-fold concentrations, respectively, of the LOQ (1.5 ppm) of PA. It should be noted here that a high selectivity was attained for PA over DNP, while the CRREL colorimetric procedure gave somewhat close response factors for TNT,

2,4-DNT and PA (i.e. TNT turned the acetone extract red, 2,4-DNT blue, and 2,6-DNT pink in the CRREL method [2]). The PA analysis in soil by the developed extractivephotometric procedure was successful in that the soilimpregnated PA could be washed with acetone–water after drying and homogenization of (white clay) soil, and determined almost quantitatively with a slight positive error. 3.3. Dinitrophenol determination The curve of A (i.e. the difference in absorbance at 400 nm of 1000 and 0 ppm imidazole-containing solutions of DNP) versus imidazole concentration is shown in Fig. 3. Since a somewhat leveling off of A with concentration was observed for 1000 ppm ligand (imidazole) concentrations in Fig. 3, the differences in absorbance at 400 nm of 1000 and 0 ppm imidazole-containing solutions of DNP and PA at varying concentrations were recorded, and these new A data were plotted as a function of analyte (DNP or PA) concentration (see Fig. 4). The individual spectra of varying concentrations of alcoholic DNP solutions mixed in equal volumes with 1000 ppm imidazole solution are shown in Fig. 5. The differences in absorbance between 1000 and 0 ppm imidazole-containing solutions of DNP and PA were recorded against analyte concentrations to give rather linear

0.55

0.50

∆A

0.45

0.40

0.35

0.30 0.00

200.00

400.00

600.00

800.00

1000.00

Imidazole Concn. (ppm) Fig. 3. A of 25 ppm DNP + varying concentrations of imidazole mixture solutions in alcohol (the difference in absorbance at 400 nm, A, of imidazole-containing and not containing solutions) as a function of imidazole concentration (ppm).

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91

2.00

∆A

1.60

1.20 PA DNP

0.80

0.40

0.00 0.00

20.00

40.00

60.00

80.00

concn. of analyte (DNP or PA) Fig. 4. A (difference in absorbance at 400 nm of 1000 and 0 ppm imidazole-containing solutions of DNP or PA) recorded as a function of the analyte (DNP or PA) concentration (ppm).

curves as shown in Fig. 4. PA between 10 and 25 ppm did not affect the quantitation of DNP by means of A versus concentration curves. For higher concentrations of PA in binary mixtures, PA could be estimated beforehand by the developed extractive-photometric procedure, and its A calculated by means of Fig. 4 so that the A due to DNP could be found from the difference, enabling its determination using the Fig. 4 plot. To confirm that PA did not interfere up to 25 ppm concentration levels, a separate mixture containing (25 ppm DNP + 25 ppm PA) was mixed in equal volumes with 1000 ppm imidazole, and A versus DNP concentration curve in Fig. 4 enabled the calculation of exactly 25 ppm DNP. The spectrophotometric method developed for DNP assay is based on the formation of an intermolecular charge-transfer complex between DNP and the electron-donor imidazole. Although a similar complex could also form with o-toluidine, imidazole better served the purpose. DNP itself gave a yellow colored solution in the medium chosen for analytical determination, however, this yellow color was intensified in the presence of an excess of the imidazole reagent. When PA was present in the original solution, it also gave a yellow color due to intramolecular charge transfer (CT), but a pronounced difference from DNP was that this intramolecular CT band was not intensified at comparatively low PA concentrations

upon reagent (imidazole) addition. Thus, the presence of DNP can be confirmed by an increase in absorbance at 400 nm of the analyte–imidazole mixture compared to that of the analyte alone, as only intermolecular CT bands—but not intramolecular CT bands—are expected to intensify upon reagent addition. This is in accordance with an earlier literature report stating that polynitro compounds such as 2,4,6-TNT, PA, trinitropyrene and tetranitropyrene did not need electron-donating ligands such as fluorene or butanone for color development, unlike dinitro-compounds, in a Et4 NOH solution of DMF [17]. This observation could be better quantified by measurement of the second derivative absorbances (2 D) at 426 nm, where a linear calibration between 10 and 75 ppm DNP was possible with the equation 2

D426 = 4.94 × 10−4 CDNP (ppm) + 5.59 × 10−4

with a linear correlation coefficient of r = 0.9995. It should noted that the slope of this line, and hence analytical sensitivity, is not sufficiently high. Naturally the quantification of DNP, listed as a secondary target analyte by the CRREL, along with the parent nitro-compound (PA) is important for monitoring contaminated land remediation studies. 3.4. Mononitrophenol detection The qualitative test with diphenylamine in sulfuric acid solution was proposed in the literature for detecting organic

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Fig. 5. The individual spectra of varying concentrations of alcoholic DNP solutes mixed in equal volumes with 1000 ppm imidazole solution.

nitrates, and this test proved to detect NP in aqueous solution producing a blue color. If PA (with Meisenheimer anion formation and IBMK extraction) and DNP (with imidazole CTC) were both shown to be absent, then a positive organic nitrate test with DPA/H2 SO4 indicated the presence of NP.

of the solvent, as compared to high costs of the CRREL and EnSysRISc methods reaching 15–20 USD per sample. In the Jenkins’ method, the water content of acetone is a critical parameter such that too little water causes reagent (solid KOH and Na2 SO3 ) solubility problems prolonging the optimal reagent contact time while too much water adversely affects both the stability and intensity of color [8,9]. An upper limit of 17.4% H2 O in the final solution is set in the Jenkins’ method, while the developed method is completely water-tolerant since color is developed in the IBMK extract. The developed method does not witness any reagent

3.5. Overview of TNT and PA methods The reaction scheme proposed for the Meisenheimer anion formation followed by extractive-photometric determination of TNT may be formulated as follows: CH3 O 2N -

2,4,6-TNT + OH

NO2

-

(Meisenheimer anion of TNT) OH NO 2

CH3 O 2N

Meisenheimer anion of TNT + CP+Br-

(IBMK)

CP+

NO2

-

-

+ Br OH NO2

(org)

IBMK, being a water-immiscible ketone, may also have added to the aromatic ring as a carbanion, resulting in the production of a Janowsky complex. Thus IBMK has replaced acetone, the volatile and increased flammability risk bearing solvent of the Jenkins’ method [8,9]. Further, the costs could be significantly reduced by distillation recovery

solubility problems as there is sufficient water in the medium. Thus the recommended method does not necessitate preliminary drying of soil samples in an oven which was reported to cause significant TNT losses in relatively contaminated samples [2], as opposed to the commercial col-

A. Üzer et al. / Analytica Chimica Acta 505 (2004) 83–93

orimetric method, EnSysRISc, requiring preliminary drying of soil to <10% moisture [2]. Both the CRREL and EnSysRISc methods are interfered by the presence of soil humic acids requiring background correction [2,8,9], as compared to the developed method unaffected by humates (as these substances are multivalently anionic at the employed high pH, and therefore not extracted into IBMK). Also, further reactions of the initially formed Meisenheimer–Janowsky anions of the Jenkins’ method to possible formation of dianions upon extended contact with reagents [8] is not a threat to the developed method, as dianions may not be extracted due to high hydration energies. The linear range and reproducibility of the developed method is much higher than those of the CRREL method, while the limit of detection (LOD) is perfectly acceptable as regards to decision making in clean-up operations of contaminated soil [8]. The developed extractive-photometric method for PA assay is similar to the one for TNT where a Meisenheimer– Janowsky anion of PA is formed in alkaline medium and extracted with CPB into IBMK. Although both CRREL and EnSysRISc methods may not differentiate the target analyte, TNT, from the dinitro-analogues (DNTs) [2], the developed method for trinitrophenol (PA) which makes use of a similar reaction is capable of differentiating this analyte from lower (e.g. dinitrophenol) analogues. The developed method for PA is much simpler, less expensive, and less tedious than the alternative field method developed by Thorne and Jenkins [16], based on a successive cycle of operations comprising ion-exchanger adsorption, washing, elution, color development, and background correction for compensating for soil humates. The wavelength of maximum absorption of the developed method is significantly red-shifted in comparison to that of the CRREL method [16], an important superiority which, when combined with the extractive-photometric method employed, shows a net advantage of removing interferences arising from soil materials like humic acids producing yellow extracts [16]. The Meisenheimer–Janowsky anions of TNT or PA formed in this work over a critical NaOH concentration, and solvent extraction of the ion-associate (i.e. Meisenheimer anion–CP cation pair) was possible over a critical CPB concentration, though an excess of the tenside caused solubility problems. Thus the concentration of reagents stated in the recommended procedure were optimal for high precision.

4. Conclusions Two simple colorimetric procedures suitable for field use were developed for TNT, PA and its potential degradation product, DNP. PA formed a stable orange-red ion-associate with NaOH and CPB extractable into IBMK, TNT formed a red extract under identical conditions as that of PA, and DNP formed a yellow colored intermolecular CT complex with imidazole. Although both assay methods were based on the measurement of the ordinary absorbances (at 490 nm

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for PA, 500 nm for TNT, and 400 nm for DNP), a second derivative measurement at 426 nm (with λ = 10 nm) was also suitable for linear calibration of DNP. Any NP present could be detected by the aid of the diphenylamine qualitative test. The developed colorimetric methods enabled simple, cheap and effective monitoring of PA and TNT (and possibly other polynitroaromatics)-contaminated sites where remediation efforts were to be rapidly evaluated by analyzing numerous samples. The developed PA and TNT assay would also pose a cheap and rapid alternative for the confirmation of analytical findings of criminological police laboratories running highly sophisticated and hyphenated chromatographic analyses. Acknowledgements The authors wish to thank the Istanbul University Research Fund for the partial support provided to this research through the project with reference number T-1214/01112001. The authors extend their gratitude to Istanbul Police Headquarters, Criminology Laboratory (Istanbul Emniyet Mudurlugu, Kriminal Polis Laboratuvari) for enabling access to the laboratory facilities and providing the TNT sample. References [1] T.F. Jenkins, M.E. Walsh, P.W. Schumacher, P.H. Miyares, C.F. Bauer, C.L. Grant, J. AOAC 72 (1989) 890. [2] A.B. Crockett, T.F. Jenkins, H.D. Craig, W.E. Sisk, US Army Corps of Engineers, Cold Regions Research & Engineering Laboratory, Special Report 98-4, February 1998. [3] C.A. Heller, S.R. Grenl, E.E. Erickson, Anal. Chem. 54 (1982) 286. [4] E.D. Erickson, D.J. Knight, D.J. Burdick, S.R. Greni, Naval Weapons Center, Report NWC TP 6569, China Lake, CA, 1984. [5] T.F. Jenkins, P.W. Schumacher, US Army Corps of Engineers, Cold Regions Research & Engineering Laboratory, Special Report 90-20, 1990. [6] S. Stevanovic, M. Mitrovic, Int. J. Environ. Anal. Chem. 40 (1990) 69. [7] Y. Zhang, W.R. Sunberg, C.L. Seitz, D.C. Grant, Anal. Chim. Acta 217 (1989) 217. [8] T.F. Jenkins, US Army Corps of Engineers, CRREL, Special Report 90-38, 1990. [9] T.F. Jenkins, M.E. Walsh, Talanta 39 (1992) 419. [10] T.F. Jenkins, M.E. Walsh, Reagent chemistry for on-site TNT/RDX determination, in: V. Lopez-Avila (Ed.), Current Protocols in Analytical Chemistry, vol. 2, Wiley, New York, Unit 2D, 1998. [11] A.D. Hewitt, T.F. Jenkins, US Army Corps of Engineers, CRREL, Special Report 99-9, 1999. [12] T.F. Jenkins, P.W. Schumacher, J.G. Mason, P.G. Thorne, US Army Corps of Engineers, CRREL, Special Report 96-10, 1996. [13] J. Von Meisenheimer, Liebig’s Annalen der Chemie 323 (1902) 205. [14] R.T. Medary, Anal. Chim. Acta 258 (1992) 341–346. [15] H.D. Craig, W.E. Sisk, M.D. Nelson, W.H. Dana, in: Proceedings of the 10th Annual Conference on Hazardous Waste Research, Great Plains Rock Mountain Hazardous Substance Research Center, Manhattan, KS, 23–24 May 1995. [16] P.G. Thorne, T.F. Jenkins, US Army Corps of Engineers, CRREL, Special Report 95-20, 1995. [17] E. Sawicki, T.W. Stanley, Anal. Chim. Acta 23 (1960) 551.