Analytica Chimica Acta 403 (2000) 145154

Synthesis, characterisation and preliminary analytical evaluation of three oxamide reagents for peroxyoxalate chemiluminescence
Neil W. Barnett , Richard Bos, Raelene N. Evans, Richard A. Russell
School of Biological and Chemical Sciences, Deakin University, Geelong, 3217 Australia Received 16 June 1999; accepted 3 August 1999

Abstract The synthesis, characterisation and preliminary analytical evaluation of three oxamide reagents, for peroxyoxalate chemiluminescence, are described. The analytical gures of merit for the three oxamides were assessed using ow injection analysis and the uorophores rhodamine B and uoroescein in a solvent mixture consisting of tetrahydrofuran, acetonitrile and aqueous buffer (1 + 1 + 2 by volume). The calibration functions approached linearity and the best detection limit (for both analytes) was 2 109 M when using the oxamide 2,2 -oxalyl-bis[(triuoromethanesulfonyl)imino]ethylene-bis(N-methylpyridinium) triuoromethanesulfonate. These results were found to compare favourably with those obtained for, the popular peroxyoxalate chemiluminescence reagent, bis(2,4,6-trichlorophenyl) oxalate (TCPO), using an identical solvent system. However, the detection limits achieved with TCPO were degraded at pH 10 due to a signicant increase in the background emission. The addition of the base catalyst, imidazole, to these chemiluminescent reactions resulted in the deterioration of analytical performance for both TCPO and the oxamide reagents. 2000 Elsevier Science B.V. All rights reserved.
Keywords: Peroxyoxalate chemiluminescence; Synthesis of oxamides; Bis(2,4,6-trichlorophenyl) oxalate; Flow injection analysis

1. Introduction The chemiluminescent system based upon the reaction between various oxalic acid derivatives and hydrogen peroxide in the presence of a suitable uorophore was rst described by Chandross [1] in 1963. Following further developments of this chemistry by Rauhut and co-workers [25], more efcient reagents became available for what is now referred to as peroxyoxalate chemiluminescence. The mechanism of the peroxyoxalate chemiluminescence reaction has been postu Corresponding author. Tel.: +61-03-5227-1291; fax: +61-03-5227-2218 E-mail address: barnie@deakin.edu.au (N.W. Barnett)

lated to involve at least one highly energetic intermediate (possibly a dioxetane species) capable of exciting a uorescent receptor molecule [6,7]. This highly efcient chemiluminescent system enables the excitation of various uorophores having emission wavelengths from the ultraviolet to near infrared, which facilitates its utilisation for numerous analytical applications [8,9]. The intrinsic background emission observed from the oxidation of peroxyoxalate reagents, in the absence of a uorophore [1016], can be a signicant problem as it may result in poor signal to noise ratios and consequently degraded detection limits. In a recent study [17], we postulated that the background emission from the reaction of diaryl oxalate esters with hydrogen peroxide was a result of chemiluminescence

0003-2670/00/$ see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 9 9 ) 0 0 6 3 9 - X

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from the oxidised phenoxide-leaving group (a phenoxyl radical), the intensity of which was increased at high pH [16,17]. Given that the most common analytical application of peroxyoxalate chemiluminescence is liquid chromatographic detection [8,9], the necessity to use organic solvents for the dissolution of the reagents such as bis(2,4,6-trichlorophenyl)oxalate (TCPO) has been shown to cause post column mixing problems when partially aqueous mobile phases are employed [18]. Solid reactors containing TCPO will tolerate an aqueous content of up to 30% (v/v) [19], but the solubility of the reagent was very low when using this approach. Since 1976, several water-soluble oxalate esters and amides have been reported, initially by Mohan and co-workers [20,21] and more recently from our own laboratory [22]. Interestingly, we found [17] that certain of these oxamide reagents [21] exhibited no measurable background emission in the absence of a uorophore. However, only two of these compounds [21] have been evaluated analytically [2330]. Therefore, reagents exhibiting good analytical performance and enhanced aqueous compatibility would be desirable. Towards this end, we have synthesised three previously described oxamides [21] and compared their analytical performance to that of TCPO. The decision to synthesise the particular reagents shown in Fig. 1 was made on the basis of their reported chemiluminescent quantum yields [21]. The effect of the base catalyst imidazole upon these aqueous peroxyoxalate chemiluminescence reagents has also been investigated.

Fig. 1. Structures of the three oxamide reagents evaluated in this study.

Fig. 2. Flow injection system employed for the analytical evaluation investigation of the peroxyoxalate chemiluminescence reagents.

2. Experimental 2.1. Instrumentation The ow injection analysis system shown schematically in Fig. 2 was assembled from a Gilson Minipuls 3 peristaltic pump (Villiers le Bel, France). The peristaltic pump tubing was bridged poly(vinyl chloride) (PVC) for aqueous solutions and silicone for solvent streams (AI Scientic, Scarborough, Queensland, Australia), connected using PTFE tubing (0.3 mm i.d.) and utilising polypropylene angeless ttings (Cole Palmer, Wantirna South, Vic., Australia). A six-port injection valve (Rheodyne 5020, Cotati, CA) was used to introduce samples into stream A. The ow cell was a spiral glass coil with the conuence point 1 cm from the beginning of the spiral (Embell Scientic, Murwillumbah, Australia). The detector comprised of a photomultiplier tube, model 9924 BS operated at 600 V, provided by a power supply (PM28BN) and a voltage divider model C611 by (Thorn-EMI, Hayes, Middlesex, UK). The photomultiplier tube and voltage divider were encased in a light-tight housing with foam padding to protect the detector unit assembly from vibration and shock. Output from the detector was recorded on a strip chart recorder (Omniscript, Houston Instruments, Austin, TX), and the response

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was measured manually using peak height. All pH measurements were made using a 120 model Corning pH meter (Corning, Halstead, Essex, UK). A 300 MHz Unity Plus nuclear magnetic resonance spectrometer (Varian, Palo Alto, CA) was used to characterise and determine the purity of the oxamide reagents synthesised. Mass spectra were recorded using a Micromass Platform (II) electrospray mass spectrometer (Micromass, Altrincham, Cheshire, UK). 2.2. Reagents The reagents used in this study were TCPO (>98% purity, Tokyo Kasai Kogyo, Japan), 4-(2-aminoethyl) morpholine (99%), 2-(2-aminoethyl)pyridine, (95%), 4-amino-1-benzylpiperidine, (>98%), methyl triuoromethanesulfonate, (>99%), triuoromethanesulfonic anhydride, rhodamine B (90%) (Aldrich Chemical Co., Milwaulkie, MI), uorescein (LR grade, Ajax Chemicals, Sydney, Australia). Tetrahydrofuran (99.7%, Hypersolv grade), acetonitrile (99%, Hypersolv grade), and hydrogen peroxide (100 volume, AR grade) were all obtained from Merck, Poole, Dorset, UK. All other reagents were of analytical reagent grade and prepared with deionised water from a Waters Milli-Q reverse osmosis system.

3. Synthesis The three oxamide reagents used in this study (see Fig. 1) were all prepared using a previously described [21] route, which is summarised below in Scheme 1. However, in our hands, the synthetic procedures published by Mohan and co-workers [21,23] afforded neither the precursors nor the target molecules in useful yields. Consequently, we have included our detailed synthesis and denitive characterisations within Section 5.

4. Analytical procedures
Scheme 1.

The ow injection system shown in Fig. 2 was congured such that stream A carried the buffered aqueous carrier solutions (at pH 8, 9 and 10). The various buffers were prepared as follows: pH 8, NaH2 PO4 /K2 HPO4 (0.01 M), pH 9 and 10,

NaHCO3 /Na2 CO3 (0.025 M). These buffers were adjusted to the desired pH with either sodium hydroxide (1 M) or hydrochloric acid (1 M) as required. Stream B contained a solution of one of the four peroxyox-

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alate reagents, TCPO or oxamides 1, 2 or 3 prepared at a concentration of 5 103 M, in a solution of (1 + 1) acetonitrile/tetrahydrofuran in the presence of hydrogen peroxide (0.7 M). The ow rates used for both streams was 2.5 ml min1 for all experiments. Three analysis cycles were carried out for each of the 10 uorescein solutions (from 5 109 to 1 104 M) and 10 rhodamine B solutions (from 5 109 to 1 104 M). For the experiments involving the addition of the base catalyst imidazole, this was made up to a concentration of 5 103 M in the carbonate buffer, and adjusted to pH 9. Three analysis cycles were then carried out for each of the seven uorescein solutions (from 5 107 to 1 104 M). 5. Results and discussion 5.1. Synthesis 5.1.1. 2,2 -Oxalyl-bis[(triuoromethanesulfonyl) imino]ethylene-bis(N-methylpyridinium) triuoromethanesulfonate (Oxamide 1) 5.1.1.1. Step 1. Triuoromethanesulfonic anhydride (5.87 g, 0.021 mol) was added drop-wise to a stirred solution of 2-(2-aminoethyl)pyridine (5 g, 0.041 mol) in dry dichloromethane (40 ml) at 0 C under a nitrogen atmosphere. After the addition was completed, the mixture was stirred at room temperature for 2 h. The resultant red/black mixture was then evaporated to give a dark oil which was mixed with cellulose powder (Whatman CF11). This mixture was placed in a cellulose thimble in a Soxhlet extractor and extracted for 2 h with cyclohexane. The resultant extract was then evaporated to give yellow crystals which were recrystallised from methylcyclohexane and washed with petroleum ether (4060 C) to give the pure white N-[2-(2 -pyridyl)ethyl]triuoromethanesulfonamide with a 95% yield. In CDCl3 , 1 H NMR 3.03 (2H, dd), 3.65 (2H, dd), 7.14 (2H, m), 7.60 (1H, t), 7.80 (1H, ex), 8.43 (1H, d), 13 C NMR 36.9, 43.4, 119.8 (q, 1 J = 322 Hz), 122.3, 123.8, 137.3, 148.7, 158.0, 19 F NMR 78.0.

very slowly to a stirred solution of N-[2-(2 -pyridyl) ethyl]triuoromethane sulfonamide (3.75 g, 0.0015 mol) and triethylamine (2.0 g, 0.02 mol) in dry tetrahydrofuran (60 ml) at 0 C under a nitrogen atmosphere. After the addition was completed, the mixture was stirred at room temperature for 24 h. The triethylammonium chloride salt was ltered off to leave the product in the ltrate. The ltrate was evaporated to give the crude product which was recrystallised from cyclohexane and washed with petroleum ether (4060 C) to give white crystals of N,N -bis[2-(2 -pyridyl)ethyl]-N,N -bis(triuoromethylsulfonyl)oxamide crystals with a yield of 65%. In CDCl3 , 1 H NMR 3.26 (2H, b), 4.15 (1H, b), 4.41 (1H, b), 7.23 (2H, m), 7.66 (1H, dd), 8.57 (1H, d), 13 C NMR 35.2, 47.4, 119.1, (q, 1 J = 324 Hz) 121.9, 123.5, 136.6, 149.5, 156.8, 159.7, 19 F NMR 73.4. 5.1.1.3. Step 3. Methyltriuoromethanesulfonate (0.72 g, 0.004 mol) was added drop-wise to a solution of the N,N -bis[2-(2 -pyridyl)ethyl]-N,N -bis(triuoromethylsulfonyl)oxamide (0.5 g, 0.0009 mol) in dry dichloromethane (30 ml) at 0 C under a nitrogen atmosphere. After the addition was completed, the reaction mixture was stirred at room temperature for 2 h. The resultant white solid was separated by ltration and washed with dichloromethane to obtain the white product (see Fig. 1, Structure 1) with a yield of 90%. In d-6 acetone, 1 H NMR 3.77 (2H, b), 4.50 (1H, b), 4.65 (3H, s), 4.75 (1H, b), 8.14 (1H, t), 8.26 (1H, d) 8.68 (1H, t), 9.15 (1H, d), 13 C NMR 45.4, 45.9, 119.1 (q, 1 J = 324 Hz), 121.2 (q, 1 J = 321 Hz), 126.8, 129.9, 145.9, 147.6, 153.4, 159.5, 19 F NMR 73.2, 78.6. 5.1.2. 4,4-Oxalyl-bis[(triuoromethylsulfonyl) imino]-bis(N-methyl-N-benzylpiperidinium) triuoromethanesulfonate (Oxamide 2) 5.1.2.1. Step 1. Triuoromethanesulfonic anhydride (3.7 g, 0.013 mol) was added drop-wise to a stirred solution of 4-amino-1-benzylpiperidine (4.2 g, 0.026 mol) in dry dichloromethane (25 ml) at 0 C, under a nitrogen atmosphere. After the anhydride had been added, the solution was stirred for a further 2 h at room temperature. The reaction mixture was then ltered to remove the salt by-product. The re-

5.1.1.2. Step 2. Oxalyl chloride (1.1 g, 0.0086 mol) in dry tetrahydrofuran (15 ml) was added drop-wise

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sultant black mixture was evaporated to give a dark resinous oil which was mixed whilst still hot with cellulose powder (Whatman CF11). This mixture was placed in a cellulose thimble in a Soxhlet extractor and extracted for 2 h with cyclohexane. The resultant extract was evaporated to give yellow crystals which were recrystallised from methylcyclohexane to give N-(N -benzyl)-4-piperidinyl-triuoromethanesulfonamide in 95% yield. In CDCl3 , 1 H NMR 1.63 (2H, m), 2.05 (2H, m), 2.13 (2H, m), 2.83 (2H, m), 3.49 (1H, m), 3.49 (1H, ex), 3.49 (2H, s), 7.30 (5H, m), 13 C NMR , 34.6, 53.2, 54.3, 63.5, 121.3, (q, 1 J = 321 Hz), 128.2, 129.4, 130.1, 139.8, 19 F NMR 78.6.

diethyl ether. The white solid product (see Fig. 1, Structure 2) was then separated by ltration and washed with diethyl ether giving a yield of 65%. In d-6 acetone, 1 H NMR 2.21 (2H, m), 2.97 (2H, m), 3.26 (1H, m), 3.26 (2H, s), 3.77 (2H, m), 3.92 (2H, m), 4.78 (3H, s), 7.49 (3H, m), 7.63 (2H, m), 13 C NMR (inverse gated), 23.0 (1C), 23.5 (1C), 44.9 (1C), 58.3 (1C), 59.9 (2C), 72.1 (1C), 120.2 (1C, q, 1 J = 324 Hz), 122.6 (1C, q, 1 J = 320 Hz), 128.4 (1C), 130.4 (2C), 132.0 (1C), 134.8 (2C), 160.8 (1C), 19 F NMR 74.1, 78.8. 5.1.3. 4,4 -Oxalyl-bis[(triuoromethylsulfonyl) imino]ethylene-bis(N-methylmorpholinium) triuoromethanesulfonate (Oxamide 3) 5.1.3.1. Step 1. Triuoromethanesulfonic anhydride (8.4 g, 0.03 mol) was added drop-wise to a stirred solution of N-(2-aminoethyl)morpholine (7.56 g, 0.06 mol) in dry dichloromethane (50 ml) at 0 C, under a nitrogen atmosphere. After the anhydride had been added, the solution was stirred for a further 2 h at room temperature. The reaction mixture was ltered to remove the salt by-product. The resultant black reaction mixture was evaporated to give a dark oil which was mixed with cellulose powder (Whatman CF11) whilst still hot. This mixture was placed in a cellulose thimble in a Soxhlet extractor and extracted for 2 h with cyclohexane. The resultant extract was then evaporated to give yellow crystals which were recrystallised from methylcyclohexane to give N-(2-morpholinoethyl)triuoromethanesulfonamide as white aky crystals with a yield of 95%. In CDCl3 , 1 H NMR , 2.52 (4H, dd), 2.61 (2H, dd), 3.39 (2H, dd), 3.74 (4H, dd), 4.50 (1H, ex), 13 C NMR 40.0, 53.0, 56.7, 66.6, 119.7, (q, 1 J = 321 Hz), 19 F NMR 78.9.

5.1.2.2. Step 2. Oxalyl chloride (1.45 g, 0.0091 mol) in dry tetrahydrofuran (15 ml) was added drop-wise to a stirred solution of N-(N -benzyl)-4-piperidinyltriuoromethane sulfonamide (6.42 g, 0.02 mol) and triethylamine (2.42 g, 0.024 mol) in dry tetrahydrofuran (20 ml) at 0 C under a nitrogen atmosphere. After the addition was completed, the mixture was stirred at room temperature for 24 h. The triethylammonium chloride salt was ltered off to leave the product in the ltrate. The ltrate was evaporated to afford the crude product which was recrystallised from cyclohexane to give white crystals of N,N-bis[(1-benzyl)-4-piperidinyl]-N,N -bis[(triuoromethyl)sulfonyl]oxamide with a yield of 65%. In CDCl3 , 1 H NMR 1.77 (2H, m), 2.05 (2H, m), 2.61 (2H, m), 3.00 (2H, m), 3.53 (2H, s), 3.97 (1H, m), 7.31 (5H, m), 13 C NMR (inverse gated), 28.1 (2C), 53.0 (2C), 62.3 (1C), 62.8 (1C), 119 (1C, q, 1 J = 324 Hz), 127.1 (1C) 128.2 (2C), 129.0 (2C), 138.0 (1C), 159.5 (1C), 19 F NMR 74.8.

5.1.2.3. Step 3. Methyltriuoromethane sulfonate (16 g, 0.098 mol) was added drop-wise to a solution of N,N-bis[(1-benzyl)-4-piperidinyl]-N,N -bis[(triuoromethyl)sulfonyl]oxamide (11.0 g, 0.016 mol) in dry dichloromethane (150 ml) at 0 C under a nitrogen atmosphere. After the addition was completed, the reaction mixture was stirred at room temperature for 4 h. Dichloromethane was removed from the brown oily solid, which was then triturated overnight with

5.1.3.2. Step 2. Oxalyl chloride (0.185 g, 0.0013 mol) in dry tetrahydrofuran (5 ml) was added drop-wise to a stirred solution of N-(2-morpholinoethyl)triuoromethanesulfonamide (0.63 g, 0.0024 mol) and triethylamine (0.26 g, 0.0025 mol) in dry tetrahydrofuran (30 ml) at 0 C under a nitrogen atmosphere. After the addition was completed, the mixture was stirred at room temperature for 24 h. The triethylammonium

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chloride salt was ltered off to leave the product in the ltrate. The ltrate was evaporated to give crude N,N -bis(2-morpholinoethyl)-N,N -bis(triuoromethylsulfonyl)oxamide which was recrystallised from cyclohexane to give a yield of 35%. In CDCl3 , 1 H NMR 3.96 (1H, b), 3.81 (1H, b), 3.68 (4H, dd), 2.66 (2H, dd), 2.52 (4H, dd), 13 C NMR 44.8, 53.6, 54.9, 66.8, 119.1, (q, 1 J = 324 Hz), 159.2, 19 F NMR 73.8.

5.1.3.3. Step 3. Methyltriuoromethanesulfonate (0.17 g, 0.001 mol) was added drop-wise to a solution of N,N -bis(2-morpholinoethyl)-N,N -bis(triuoromethylsulfonyl)oxamide (0.25 g, 4.3 104 mol) in dry dichloromethane (10 ml) at 0 C under a nitrogen atmosphere. After the addition was completed, the reaction mixture was stirred at room temperature for 2 h. The resultant white solid was separated by ltration and washed with dichloromethane to obtain the product (see Fig. 1, Structure 3) with a yield of 55%. In d-6 acetone, 1 H NMR 3.66 (3H, s), 3.92 (4H, dd), 4.05 (2H, m), 4.12 (2H, dd), 4.22 (2H, m), 4.70 (1H, b), 4.90 (1H, b), 13 C NMR 40.4, 46.6, 59.8, 60.3, 60.5, 118.9 (q, 1 J = 324 Hz), 121.3 (q, 1 J = 321 Hz), 158.8, 19 F NMR 73.2, 78.9. Our initial efforts to follow the original synthetic procedures [21,23] as summarised in Scheme 1 were unsuccessful. We found that isolation of the triyamides (after Step 1) from the resultant dark oils was all but impossible using the recommended [21,23] recrystallisation. However, change to a Soxhlet extractor efciently overcame these problems. Also, the prescribed [21,23] reaction times for Step 2 (see Scheme 1) were typically 24 h, which proved to be inadequate and required considerable extension (up to 24 h) so as to afford reasonable yields of the unquaternised oxamides. The melting point data for each of the oxamide reagents and their respective precursors is listed in Table 1. With the exception of the melting point for the product from Step 2 of the synthesis of oxamide 2, agreement between our values and those of Mohan et al. [21] and Tseng and Rauhut [23] was quite good for the other ve synthons. It should be noted that the target compound from Step 2 of the synthesis of oxamide 2 was difcult to obtain in high purity, and we also recorded a melting point which ranged from

60 to 65 C for one of our less pure products. However, without exception, the literature [21,23] values for oxamides 1, 2 and 3, are, in each case, considerably lower than those observed in this study. Interestingly, Grayeski et al. [26] reported a melting point for oxamide 3 which is in excellent agreement with that shown in Table 1. The NMR data listed previously is entirely consistent with the structures of the precursors (see Scheme 1), and the nal products (see Fig. 1). The evidence for the conrmation of the structures of the three oxamide reagents is further supported by the electrospray mass spectral data which is summarised in Table 2. Consequently, we are condent that the three peroxyoxalate chemiluminescence reagents that we have evaluated for analytical performance were those compounds whose structures are shown in Fig. 1.

5.2. Analytical evaluation The analytical gures of merit obtained for the three oxamides and TCPO in conjunction with the two uorphores, rhodamine B and uorescein, are summarised in Tables 3 and 4, respectively. From this data, it can be seen that the performance of both oxamide 1 and oxamide 3 was independent of the uorophore and either equal to or slightly superior than that achieved using TCPO at pH 8 and pH 9. However, in the case of the oxamide 2, the detection limits were consistently poorer, by approximately an order of magnitude, for each of the three pH values irrespective of the uorophore employed. Mohan et al. [21] found that the chemiluminescence quantum efciency of oxamide 2 (0.045), in an aqueous micellar system, was slightly less than that for either oxamide 1 or oxamide 3 (0.072 and 0.048, respectively). It is important to note that the original report [21] sought to evaluate these compounds with a view to establishing their performance as long-term chemiluminescent light sources operating under predominantly aqueous conditions. Therefore, the generally inferior analytical gures of merit achieved with oxamide 2 in the current investigation are not completely attributable to the differences in the measured quantum efciencies for these reagents [21]. Grayeski et al. [26] observed that, when using oxamide 3 over the pH range 411 in a static system, an increase in the pH realised an increase in

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Table 1 Comparison of the melting point data obtained in the present study for all target compounds and synthons with values obtained from previous works Synthesis Step Melting points ( C) This study Oxamide 1 1 2 3 1 2 3 1 2 3 83.585.5 133135 193195 127128 133134 198211a 105107 7376 193194 Literature values 8385 [21] 136138 [21] 173178 [21], 162165 [23] 124126 [21] 130133 [21] 125130 [21] 106108 [21,23] 6264 [21,23] 160165 [21], 121125 [23], 195197 [26]

Oxamide 2

Oxamide 3

There was considerable decomposition observed at the melting point.

Table 2 Comparison of the calculated and expected electrospray mass spectral data of the three oxamide reagents Compound Molecular ion mass Cation (m/z = 2) Measured Oxamide 1 Oxamide 2 Oxamide 3 296.1 364.1 304.0 Calculated 296.23 364.34 304.18 Anion (m/z = 1) Measured 148.9 148.9 148.8 Calculated 149.02 149.02 149.02

Table 3 Analytical gures of merit for compounds 1, 2, 3 and TCPO in (1 + 1 + 2 by volume) tetrahydrofuran/acetonitrile/water using uoroscein as the uorophore Fluorescein data Reagent Oxamide 1 pH 8 9 10 8 9 10 8 9 10 8 9 10 Linear/linear data Range (M) 5 109 5 106 5 109 5 106 5 109 1 105 5 108 5 106 5 108 5 105 5 108 1 105 5 109 5 106 5 109 5 106 5 109 1 105 5 109 5 106 5 109 5 106 2 107 1 105 Calibration function 7 109 x + 131.19 5 109 x 21.597 3 109 x 10.433 4 109 x 38.445 3 109 x 127.92 1 109 x 92.721 4 109 x 80.616 1 109 x + 16.29 3 109 x 93.702 7 109 x 18.258 4 109 x 62.857 2 109 x + 19.274 r2a 0.9991 0.9999 0.9999 0.9999 0.9987 0.9998 0.9997 0.9994 0.9999 1.0000 1.0000 0.9999 DLb 3 109 2 109 5 109 3 108 3 108 2 108 4 109 8 109 5 109 2 109 2 109 2 107 Log/log data Range (M) 1 108 5 105 5 108 5 105 5 108 5 105 1 108 5 105 5 108 5 105 5 107 1 105 1 108 5 105 5 108 1 105 1 108 1 105 1 108 5 105 1 108 1 105 2 107 5 105 Calibration function 1.0949x + 10.408 1.0024x + 9.7318 1.0661x + 9.8653 1.0022x + 9.5556 1.0447x + 9.6199 1.0804x + 9.5878 1.0283x + 9.7402 1.0022x + 9.1398 1.0085x + 9.4682 1.0565x + 10.135 1.0368x + 9.7830 0.8865x + 8.6892 r2a 0.9940 0.9986 0.9993 0.9973 0.9998 0.9988 0.9972 0.9975 0.9939 0.9991 0.9964 0.9882

Oxamide 2

Oxamide 3

TCPO

a b

n = 6. Detection limit, dened as three times the peak-to-peak baseline noise.

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Table 4 Analytical gures of merit for compounds 1, 2, 3 and TCPO in (1 + 1 + 2) tetrahydrofuran/acetonitrile/water using rhodamine B as the uorophore Rhodamine B data Reagent Oxamide 1 pH 8 9 10 8 9 10 8 9 10 8 9 10 Linear/linear plot data Range (M) 5 109 1 106 5 109 5 106 5 109 5 106 5 108 5 106 5 108 1 105 1 108 1 105 5 109 1 106 5 109 1 105 5 109 5 106 5 109 5 106 5 109 1 105 5 108 1 105 Calibration function 3 1010 x 162.44 7 109 x + 89.568 6 109 x 235.12 6 109 x 345.21 2 109 x 68.239 2 109 x 186.44 1 1010 x + 89.976 8 108 x + 40.601 5 109 x 206.49 5 109 x 124.08 9 108 x 91.548 3 109 x + 66.298 r2a 0.9984 0.9999 0.9988 0.9995 0.9995 0.9953 0.9956 0.9996 0.9984 0.9997 0.9934 0.9962 DLb 2 109 2 109 2 109 5 108 3 108 1 108 2 109 3 109 2 109 3 109 3 109 3 108 Log/log plot data Range (M) 1 108 5 105 5 107 5 105 1 108 5 105 5 107 5 105 5 107 5 105 5 107 5 105 1 108 5 105 5 107 5 105 1 108 5 105 1 108 1 105 5 107 5 105 1 108 1 105 Calibration function 0.9915x + 9.5945 0.9960x + 9.8072 0.9606x + 9.5103 1.0115x + 9.8246 1.0135x + 9.2116 1.0175x + 9.1625 0.9649x + 9.8046 0.9344x + 8.5698 0.9915x + 9.5945 0.9885x + 9.5873 0.9826x + 8.7149 1.0224x + 9.5143 r2a 0.9949 0.9980 0.9903 0.9985 0.9951 0.9954 0.9825 0.9982 0.9949 0.9929 0.9903 0.9965

Oxamide 2

Oxamide 3

TCPO

a b

n = 6. Detection limit, dened as three times the peak-to-peak baseline noise.

Table 5 Analytical gures of merit for compounds 1, 2, 3 and TCPO in (1 + 1 + 2) tetrahydrofuran/acetonitrile/water using uoroscein as the uorophore in the presence of 5 104 M imidazole Imidazole data Reagent Oxamide 1 Oxamide 2 Oxamide 3 TCPO
a b

Linear/linear data Range (M) 5 107 1 104 5 107 1 104 5 107 1 104 5 107 1 104 Calibration function 5 107 x + 6.7071 2 107 x 11.097 2 107 x 8.7652 1 108 x + 20.992 r2a 0.9990 0.9960 0.9991 0.9998 DLb 5 107 3 107 4 107 3 107

Log/log data Range (M) 5 107 1 104 1 107 1 104 5 107 1 104 1 107 1 104 Log/log calibration 1.0083x + 8.7575 0.9653x + 7.1791 0.9348x + 7.0729 0.9818x + 7.9108 r2a 0.9970 0.9985 0.9931 0.9998

pH 9 9 9 9

n = 6. Detection limit, dened as three times the peak-to-peak baseline noise.

the rate of the reaction, and improvements in the intensity of chemiluminescence emission for the uorophores rhodamine B and aminopyrenesulfonic acid. The emission intensity was observed to double over the pH range 810, where the maximum signal was obtained. However, we observed only subtle variations in the detection limits with changes in basicity for each of the three oxamides in the presence of either uorophore. When restricted to appropriate concentration ranges, the log/log calibration functions (see Tables 3 and 4) closely approximated to linearity, with the majority exhibiting slopes of 1.00 0.05 over two to three orders of magnitude. The most signicant departure from linearity was recorded from the interaction

of TCPO and uorescein at pH 10, which was also accompanied by a marked deterioration in the detection limits compared to those obtained with oxamide 1 and oxamide 3 under identical reaction conditions (see Tables 3 and 4). Interestingly, both calibration function linearity and detection limit were more adversely affected for the TCPO/uorescein combination than for the corresponding experiment with rhodamine B at the highest pH. As noted above, there was no signicant degradation of the response from the oxamide reagents, which paralleled the behaviour with TCPO. In an earlier study [17], we postulated that the background emission observed with substituted diaryloxalate reagents was caused by the generation of phe-

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Fig. 3. Comparison of the signal characteristics obtained for TCPO and oxamide 1 at pH 10 using a 1 107 M uorescein standard in (1 + 1 + 2) tetrahydrofuran/acetonitrile/water; note the difference in the Y scale for each reagent.

noxyl radicals, and that such species could not be formed with either oxamide 1 or oxamide 2 under conditions similar to those used in the present work. As can be seen from Fig. 3, the chemiluminescence signal intensity monitored from the interaction of TCPO with a uorescein standard solution (1 107 M) is approximately two and a half times that achieved using oxamide 1 with the same uorophore under identical reaction conditions. However, the relatively high background noise level associated with the peaks generated using TCPO gave a signal to noise ratio of only 2 : 1, whereas those obtained from oxamide 1 were in excess of 50 : 1. Consequently, the detection limits listed in Tables 3 and 4 (at pH 10) illustrate conclusively that the superior signal to noise ratio attained with oxamide 1 more than compensated for the reduction in response. Similar signal to noise ratios were also observed for the other combinations of oxamides and uorophores. The effect of the inclusion of the base catalyst imidazole (5 104 M at pH 9) into stream B (see Fig. 2) was examined for all four reagents with uorescein as the analyte and the results of these experiments are summarised in Table 5. The addition of this particular catalyst has been demonstrated previously to signicantly enhance the signal intensity in certain peroxyoxalate chemiluminescence systems [31], due to the formation of the highly reactive intermediate

1,1 -oxalyldiimidazole [32,33]. This species is thought to react more rapidly than the diaryloxalate reagent to generate intense chemiluminescence exhibiting good signal to noise characteristics [31]. As can be seen from Table 5, the addition of imidazole was found, without exception, to result in a marked degradation in the detection limits compared to the corresponding imidazole free results shown in Table 3. The deterioration in detectability observed during the present investigation most probably results from an initial imidazole mediated decomposition of the reagent to form 1,1 -oxalyldiimidazole [32,33], followed by rapid hydrolysis of this highly reactive intermediate in the highly basic aqueous environment [34]. In a recent paper, Appelblad et al. [35], used 1,1 -oxalyldiimidazole as a post column peroxyoxalate chemiluminescence reagent for the detection of C-21 ketosteroids following separation by liquid chromatography. In their work, a special detector was constructed to minimise the hydrolysis of 1,1 -oxalyldiimidazole following the mixing of the anhydrous 1,1 -oxalyldiimidazole stream with the column efuent [35]. Presumably, the distance from the point of conuence of stream A and stream B (see Fig. 2) to the detector (10 mm) coupled with the basic conditions employed, would appear to have been sufcient to facilitate the hydrolysis of 1,1 -oxalyldiimidazole [35]. This in turn resulted in an increase in the detection limits through the lowering of the available reagent concentration. Further evaluation of the efcacy of the base catalyst imidazole, in conjunction with these and other [22] aqueous peroxyoxalate reagents, is currently being undertaken employing a purpose-built reaction cell; the results of this work will be reported in due course. We have, for the rst time, denitively synthesised and characterised three previously reported oxamides [21,23] and compared their analytical gures of merit and aqueous compatibility with that of the most commonly used peroxyoxalate chemiluminescence reagent, TCPO. Oxamide 1 and oxamide 3 exhibited superior analytical performance at pH 10, with respect to that achieved using TCPO. This feature offers potential application to the determination of those uorophores whose detection chemistry may be limited to, or enhanced, by more alkaline reaction conditions. Whilst the oxamides prepared demonstrated limited water solubility and stability [22,26], their struc-

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