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Determination of humic acid in natural waters by flow injection analysis with chemiluminescence detection
Analytica Chimica Acta 438 (2001) 143–148
Determination of humic acid in natural waters by flow injection analysis with chemiluminescence detection J. Michałowski∗ , P. Hałaburda, A. Kojło Institute of Chemistry, University of Białystok, al. J. Piłsudskiego 11/4, 15-443 Białystok, Poland Received 11 July 2000; received in revised form 15 November 2000; accepted 5 December 2000
Abstract A fast and sensitive direct chemiluminescence flow injection procedure for determination of humic acid in natural waters was developed. N-bromosuccinimide (NBS) after on-line hydrolysis was used as a reagent evolving chemiluminescence by the oxidation of humic acid. Use of glycine as a sensitizer enhances the signal magnitude about 100 times. The detection limit is 0.012 mg l−1 and 180 samples per hour can be determined. Almost all substances occurring in typical fresh water at normal level do not disturb the determination. Only phenols with one hydroxyl group are serious interferents, which were removed by evaporation. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Humic acid; Chemiluminescence; N-bromosuccinimide; Flow injection analysis
1. Introduction Among many organic natural compounds occurring in natural waters, humic acid is one of the predominant species. This substance is known to be the precursor of some carcinogenic compounds formed during disinfection of drinking water with chlorine [1]. Another reason for its potential importance is its ability to mobilize, transport, and immobilize organic substances like pesticides, and its activity as a natural complexion agent of toxic metals. This process can increase solubility of such metal ions [2]. Generally, a direct determination of humic acid is limited by the relatively high detection limits of existing spectrophotometric [3–5] and fluorescent procedures [6,7]. An additional problem is caused by the presence of iron in most natural waters as a serious interferent in ∗ Corresponding author. Tel.: +48-85-745-75-86; fax: +48-85-745-75-81. E-mail address: [email protected] (J. Michałowski).
spectrophotometric determinations [8,9]. This problem can be solved by the use of a column filled with DEAE-cellulose [10] or non-ionic XAD-2 resin [11], but this lengthens considerably the procedure of determination. Other methods used for the determination of humic acid are: electrochemical [12], chemiluminescent with the use of potassium permanganate in alkaline solution as an oxidant [13] and immunochemical [14]. Flow injection chemiluminescence determination of humic acid was also described [15]. Oxidizing properties of N-bromosuccinimide (NBS) are attributed to hypobromous acid which is produced by its hydrolysis [16]. Chemiluminescence produced by oxidation of some organic substances after alkaline hydrolysis of NBS was utilized by flow injection determinations of isoniazid [17], dihydralazine [18], tetracyclines [19], ammonium ion in fertilizers [20], amiloride [21], hydrazine [22] and pyrogallol [23]. A great advantage of the use of NBS instead of hypobromite is relatively good stability of this reagent.
0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 0 ) 0 1 3 6 8 - 4
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J. Michałowski et al. / Analytica Chimica Acta 438 (2001) 143–148
This paper describes the development of a fast, sensitive and selective direct chemiluminescence procedure for the determination of humic acid based on oxidation with NBS after its on-line hydrolysis. The presence of glycine in the sample causes great enhancement of chemiluminescence emission.
a coiled PTFE tube of 1 mm i.d. (length of 25 cm in six windings). The photomultiplier was operated at 1200 V, and the detector response was recorded on a 386-series personal computer with KSP software. The flow system was made of PTFE tubing of 0.8 mm i.d. The reagent and carrier streams were merged in a Perspex T-piece.
2. Experimental
2.3. Procedures
2.1. Reagents
The sample solution before injection was filtered with the use of 0.45 m pore glass filter and injected into the manifold after addition of sensitise. Solutions used for calibration were prepared by mixing appropriate amount of stock humic acid solution (100 mg l−1 ) and stock glycine solution (1000 mg l−1 ) in a volumetric flask and diluting the mixture to 100 ml with water.
Humic acid (sodium salt, technical grade) was purchased from Aldrich and used without further purification. Stock solution (100 mg l−1 ) of this salt was prepared daily by dissolving 10 mg of it in 100 ml of bi-distilled water with the use of ultrasonic bath. All other reagents used were of analytical grade and were obtained from POCh-Gliwice, Poland or Aldrich (NBS). The solution of NBS (0.04 mol l−1 ) was prepared daily by dissolving 3.558 g of this substance in 0.5 l of bi-distilled water. Phenols and organic amines solutions were prepared daily, but stock solutions (1000 mg l−1 ) of glycerine, rhodamine B, rhodamine 6G, dichlorofluorescein and calcein were stable for few weeks at room temperature. 2.2. Apparatus The flow injection set-up, shown schematically in Fig. 1, consisted of an Ismatec MS-Reglo peristaltic pump, a Model 5021 rotary injection valve (Rheodyne, Cotati, CA), and flow luminometer (KSP, Poland) with
3. Results and discussion 3.1. Optimization of parameters 3.1.1. Concentration of sodium hydroxide The influence of the concentration of sodium hydroxide in a carrier solution was investigated in the range of 0.25–3 mol l−1 . The maximum response was observed for 2 mol l−1 solution of sodium hydroxide and this concentration was chosen as the optimal one (Fig. 2A). The reproducibility was also worse at the concentration of 3 mol l−1 , which is connected with the increased viscosity of the carrier stream.
Fig. 1. Flow injection manifold used for determination of humic acid in natural waters: C: 2 mol l−1 sodium hydroxide solution, R: 0.04 mol l−1 NBS solution, P: peristaltic pump, S: injection valve, F: flow cell, L: luminometer, PC: personal computer, W: waste; distance between merging point and flow-cell was 10 cm.
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Fig. 2. Optimization of the parameters: A: sodium hydroxide concentration, B: NBS concentration, C: pumping velocity, D: sample volume.
3.1.2. Concentration of NBS Concentration of NBS in the reagent solution is an important factor influencing signal magnitude. The increase of the signal was observed until 0.05 mol l−1 (Fig. 2B), which is the limit of solubility at room temperature. At this concentration, poor reproducibility was the limiting factor and the concentration of 0.04 mol l−1 was used in further experiments. Other reason for choosing the concentration of 0.04 mol l−1 was saving of the reagent. 3.1.3. Pumping velocity The chemiluminescence was found to increase constantly with increasing pumping velocity, due to high speed of the chemiluminescent reaction. The pumping speed higher than 5.9 ml min−1 caused poor reproducibility, so this flow rate was chosen for both streams (Fig. 2C). At a high pumping velocity the flow becomes more turbulent and irreproducible, so our choice was the compromise between the peak height and reproducibility. 3.1.4. Sample volume An increase of the signal magnitude while increasing the sample volume was observed (Fig. 2D). By the sample volume higher than 400 l double peaks were recorded, so this volume was considered to be
optimal one. Splitting of the peaks was caused by the differences in the reaction velocity in the middle of the sample zone and at its edges, which often happens when the sample volume is too high. 3.1.5. Temperature Increasing the temperature of the carrier and reagent solutions does not influence the signal magnitude in a considerable degree. The slope of the calibration graph was very low at the temperature 30◦ C and higher, so the room temperature (18–20◦ C) was considered as optimal for the determination. 3.2. Parameters of the optimized method A calibration graph made with the use of commercial available humic acid salt was linear in a wide concentration range. Two working ranges of calibration were established: 0.012–1 and 1–2 mg l−1 . All calibration parameters of the optimized system are collected in the Table 1. The maximal sample throughput in the optimized system was 180 h−1 . 3.3. Effect of sensitisers An addition of different fluorofores like rhodamineB, rhodamine-6G, 3,5-dichlorofluorescein or calcein
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Table 1 Calibration parameters of the optimised system depicted in Fig. 1 Parameter
Value
Calibration range
0.012–1.0 mg l−1 , y = 0.017x + 0.6907, r 2 = 0.9909 1.0–2.0 mg l−1 , y = 17.714x + 4.2276, r 2 = 0.9953 0.012 mg l−1 3.2% (0.02 mg l−1 ) 1.2% (1.0 mg l−1 )
Limit of detection R.S.D. (n = 15)
to the sample resulted in no signal enhancement. Compounds used as sensitisers in other procedures utilizing NBS, like ammonium [17–19] or hydroxylammonium hydrochloride [23] do not enhance chemiliminescence resulting from the oxidation of humic acid. Great signal enhancement (about 100-fold) was achieved after the addition of glycine (aminoacetic acid) to the sample. The signal obtained for the mixture of humic acid and glycine is considerably higher than the sum of signals for both compounds separately. The optimal concentration of the sensitise was found 35 mg l−1 . A further increase of the concentration of glycine in the sample resulted in no signal enhancement. 3.3.1. Mechanism of the signal enhancement Among many amino compounds like ammonium, hydrazine, hydroksylamine hydrochloride, ethanolamine, n-buthylamine and glycine, only the last one produces signal enhancement in the oxidation reaction of humic acid. Because it is unlikely that glycine acts as simple fluorophore, it was supposed that it reacts with some functional group(s) of humic acid and product(s) of this reaction give rise to the signal magnitude. The most important, from the analytical point of view, functional groups of humic acid are: phenolic hydroxyl, carboxylic, carbonyl and quinone groups [24]. Many model compounds with functional groups present in humic acid particles were tested for chemiluminescent reaction with glycine. Signals of analytical importance (S/N > 3) were observed for 10 mg l−1 solutions of phenol, o-nitrophenol, resorcinol, hydroquinone, pyrogallol, but without its enhancement after the addition of glycine at 35 mg l−1 . No signals were observed for 10 mg l−1 solutions of benzoic and salicylic acids and for benzoquinone solution of the same concentration. After the addition of glycine to them, only small signal characteristic of
this substance was detected. The conclusion is that the sensitising effect cannot be attributed to the presence of phenolic, quinone or carboxylic functional groups in humic acid particles. In the same way, groups containing sulphur, like in thiophenol, 1,2-ethanothiol and thiourea were tested with negative results. Aromatic carbonyl groups were found to react with glycine, and product(s) of this condensation reaction is supposed to affect the mechanism of chemiluminescence production. An addition of glycine at concentration of 35 mg l−1 to the solution of salicylic aldehyde, benzaldehyde, p-diethylaminobenzaldehyde or p-phtalaldehyde (10 mg l−1 ) gives two to five-fold signal enhancement compared with the sum of the signals for glycine and aldehyde in separated solutions. The highest signal enhancement, five-times, was achieved for salicylic aldehyde. A reaction between amino acids and o-phtalaldehyde in the alkaline medium was used for derivation of them, which enables its sensitive fluorimetric determination [25]. Strong fluorescence of the product of this reaction suggests that its chemiluminescent activity is possible. 3.4. Study of interferences Organic and inorganic compounds typically occurring in natural waters were studied as potential interferents. Each substance was considered not to interfere if it caused a relative error less than 5% for the determination of 0.5 mg l−1 of humic acid. The results are shown in Table 2. Most of the compounds at the concentration typical of natural waters do not interfere by the determination. Only phenols with one hydroxyl group should be considered as serious interferents, which affect the signal magnitude in the concentration higher than 0.075 mg l−1 . Because these compounds are volatile, they could be removed by evaporation.
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Table 2 Study of interferences with the determination of 0.5 mg l−1 of humic acid in the proposed method Interferent
Maximum tolerated concentration (mg l−1 )
Relative error (%)
Na+
1000.0 800.0 80.0 80.0 8.0 5.0 2.0 150.0 5.0 0.5 20.0 200.0 500.0 1000.0 200.0 0.5 800.0 1000.0 0.5 5.0 1.0 5.0 5.0 0.2 5.0 2.0 15.0 20.0 0.075 0.05 0.05
−4.9 −2.4 −1.3 2.2 −1.7 −4.9 −3.5 −2.7 −2.2 −0.8 2.3 −3.5 4.9 0.2 3.9 1.3 1.7 −4.9 −0.2 3.4 −4.7 −0.6 −4.4 −4.9 −1.2 −4.3 −1.0 −2.3 −0.2 −4.7 −5.0
K+ NH4+ Ca2+ Mg2+ Fe2+ Fe3+ Zn2+ Cu2+ Cr3+ CrO4 2− Cl− CO3 2− SO4 2− SO3 2− S2− PO4 3− NO3 − NO2 − Hydroxylamine Hydrazine 2,4-Diphenylhydrazine Diphenylamine p-Nitroaniline Pyrogallol Pyrocatechol Resorcinol Hydroquinone Phenol p-Cresol 2,4,6-Trichlorophenol
For this purpose 100 ml of the sample was gently boiled until its volume was reduced to about 60 ml. This solution was transferred after cooling to the 100 ml volumetric flask, appropriate volume of the sensitise was added, and it was adjusted to the mark with bi-distilled water. The validity of this procedure of interference removal was established for solutions containing 1 mg l−1 of humic acid and 1 mg l−1 of phenol, p-cresol or phenol + p-cresol. In each case a relative error of determination was less than 1%. 3.5. Analysis of real samples In order to perform determination of humic acid in real samples standard of this substance was
prepared according to Thurman and Malcolm [26]. For this purpose, humic acid was extracted from the river water of Czarna river, which is located in Bialystok’s forest area (15 km from Bialystok). The determination of humic acid using obtained standard was performed with both, proposed and spectrophotometric [27] methods in the water samples collected from the same river. The results of determination are shown in the Table 3. Parameters of the calibration with the prepared standard were very similar to those for humic acid purchased by Aldrich: equation y = 16.157x + 2.337 for the calibration graph in the range 1–2 mg l−1 , r 2 = 0.9965, R.S.D. (n = 15) 3.4 % at the concentration 0.02 mg l−1 and the detection limit 0.014 mg l−1 .
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Table 3 Comparison of the results of determination of humic acid in real samples with the proposed and spectrophotometric methods; humic acid standard was prepared according to [26]a Sample
River 1 River 2 River 3 a
Concentration of humic acid (mg l−1 ) Proposed method (main value ± S.D.)
Spectrophotometric method [27] (main value ± S.D.) (%)
Relative error (%)
6.4 ± 0.2 5.9 ± 0.1 5.7 ± 0.1
6.6 ± 0.3–3.0 5.8 ± 0.3 5.8 ± 0.2
−3.0 1.7 −1.7
Humic acid was extracted from the river water of Czarna river.
4. Conclusions The proposed method is fast and simple. It does not require sophisticated reagents and equipment, and the only sample preparation is the addition of an appropriate amount of the sensitise to the sample solution. Sensitivity of this method is considerably better than the sensitivities of spectrophotometric procedures. Because of the uncertain nature of humic acid, the use of different calibration standards leads to differences in results of determination. The use of chemiluminescence detection for determination of humic acid improves accuracy of the procedure in comparison with spectrophotometric method, where molar absorptivities for humic acids from different sources show differences up to 273% [14,27]. References [1] E. Yamada, T. Ozeki, M. Kimura, Anal. Sci. 14 (1998) 327. [2] M. Hiraide, Anal. Sci. 8 (1992) 453. [3] G.L. Picard, G.T. Felbeck Jr., Geochim. Cosmochim. Acta 40 (1976) 1347. [4] E. Tipping, Geochim. Cosmochim. Acta 45 (1981) 191. [5] M. Hiraide, M. Ishii, A. Mizuike, Anal. Sci. 4 (1988) 605. [6] T. Almegren, B. Josefsson, G. Nyquist, Anal. Chim. Acta 78 (1975) 411. [7] G.L. Brun, D.L.D. Milbrun, Anal. Lett. 10 (1977) 1209. [8] P.D. Carpenter, J.D. Smith, Anal. Chim. Acta 159 (1984) 299.
[9] K. Ohzeki, M. Tatehana, I. Nukatsuka, R. Ishida, Analyst 116 (1991) 199. [10] T. Nomizu, M. Sanji, M. Hiraide, A. Mizuika, Anal. Sci. 5 (1989) 363. [11] M.-H. Sorouradin, M. Hirade, H. Kawaguchi, Anal. Sci. 9 (1993) 863. [12] A. Cominoli, J. Buffle, W. Haerdi, J. Electroanal. Chem. 110 (1980) 259. [13] D.F. Marino, J.D. Ingle Jr., Anal. Chim. Acta 124 (1981) 23. [14] P. Ulrich, M.G. Weller, D. Knopp, R. Niessner, Anal. Sci. 9 (1993) 795. [15] X. Hu, M. Kitano, N. Takenaka, H. Bandow, Y. Maeda, D. Zhang, Bunseki Kagaku 43 (1994) 1077. [16] S.A. Halvatzis, M.M. Timotheou-Potamia, C.E. Efstathiou, Talanta 40 (1993) 1213. [17] S.A. Halvatzis, M.M. Timotheou-Potamia, A.C. Calokerinos, Analyst 115 (1990) 1229. [18] S.A. Halvatzis, M. M Timotheou-Potamia, T.P. Hadjiioannou, Anal. Chim. Acta 272 (1993) 251. [19] S.A. Halvatzis, M.M. Timotheou-Potamia, A.C. Calokerinos, Analyst 118 (1993) 633. [20] S.A. Halvatzis, M.M. Timotheou-Potamia, Talanta 40 (1993) 1245. [21] S.A. Halvatzis, A.M. Mihalatos, L.P. Palilis, A.C. Calokerinos, Anal. Chim. Acta 290 (1994) 172. [22] A. Safavi, M.R. Baezzat, Anal. Chim. Acta 358 (1998) 121. [23] A. Safavi, M.R. Baezzat, Anal. Chim. Acta 368 (1998) 113. [24] A.H. Gillam, J.P. Riley, Anal. Chim. Acta 141 (1982) 287. [25] M. Roth, Anal. Chem. 43 (1971) 880. [26] E.M. Thurman, R.L. Malcolm, Environ. Sci. Technol. 15 (1981) 463. [27] M. Schnitzer, S.U. Khan, Humic Substances in the Environment, Mercel Dekker, New York, 1972.
Determination of humic acid in natural waters by flow injection analysis with chemiluminescence detection J. Michałowski∗ , P. Hałaburda, A. Kojło Institute of Chemistry, University of Białystok, al. J. Piłsudskiego 11/4, 15-443 Białystok, Poland Received 11 July 2000; received in revised form 15 November 2000; accepted 5 December 2000
Abstract A fast and sensitive direct chemiluminescence flow injection procedure for determination of humic acid in natural waters was developed. N-bromosuccinimide (NBS) after on-line hydrolysis was used as a reagent evolving chemiluminescence by the oxidation of humic acid. Use of glycine as a sensitizer enhances the signal magnitude about 100 times. The detection limit is 0.012 mg l−1 and 180 samples per hour can be determined. Almost all substances occurring in typical fresh water at normal level do not disturb the determination. Only phenols with one hydroxyl group are serious interferents, which were removed by evaporation. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Humic acid; Chemiluminescence; N-bromosuccinimide; Flow injection analysis
1. Introduction Among many organic natural compounds occurring in natural waters, humic acid is one of the predominant species. This substance is known to be the precursor of some carcinogenic compounds formed during disinfection of drinking water with chlorine [1]. Another reason for its potential importance is its ability to mobilize, transport, and immobilize organic substances like pesticides, and its activity as a natural complexion agent of toxic metals. This process can increase solubility of such metal ions [2]. Generally, a direct determination of humic acid is limited by the relatively high detection limits of existing spectrophotometric [3–5] and fluorescent procedures [6,7]. An additional problem is caused by the presence of iron in most natural waters as a serious interferent in ∗ Corresponding author. Tel.: +48-85-745-75-86; fax: +48-85-745-75-81. E-mail address: [email protected] (J. Michałowski).
spectrophotometric determinations [8,9]. This problem can be solved by the use of a column filled with DEAE-cellulose [10] or non-ionic XAD-2 resin [11], but this lengthens considerably the procedure of determination. Other methods used for the determination of humic acid are: electrochemical [12], chemiluminescent with the use of potassium permanganate in alkaline solution as an oxidant [13] and immunochemical [14]. Flow injection chemiluminescence determination of humic acid was also described [15]. Oxidizing properties of N-bromosuccinimide (NBS) are attributed to hypobromous acid which is produced by its hydrolysis [16]. Chemiluminescence produced by oxidation of some organic substances after alkaline hydrolysis of NBS was utilized by flow injection determinations of isoniazid [17], dihydralazine [18], tetracyclines [19], ammonium ion in fertilizers [20], amiloride [21], hydrazine [22] and pyrogallol [23]. A great advantage of the use of NBS instead of hypobromite is relatively good stability of this reagent.
0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 0 ) 0 1 3 6 8 - 4
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J. Michałowski et al. / Analytica Chimica Acta 438 (2001) 143–148
This paper describes the development of a fast, sensitive and selective direct chemiluminescence procedure for the determination of humic acid based on oxidation with NBS after its on-line hydrolysis. The presence of glycine in the sample causes great enhancement of chemiluminescence emission.
a coiled PTFE tube of 1 mm i.d. (length of 25 cm in six windings). The photomultiplier was operated at 1200 V, and the detector response was recorded on a 386-series personal computer with KSP software. The flow system was made of PTFE tubing of 0.8 mm i.d. The reagent and carrier streams were merged in a Perspex T-piece.
2. Experimental
2.3. Procedures
2.1. Reagents
The sample solution before injection was filtered with the use of 0.45 m pore glass filter and injected into the manifold after addition of sensitise. Solutions used for calibration were prepared by mixing appropriate amount of stock humic acid solution (100 mg l−1 ) and stock glycine solution (1000 mg l−1 ) in a volumetric flask and diluting the mixture to 100 ml with water.
Humic acid (sodium salt, technical grade) was purchased from Aldrich and used without further purification. Stock solution (100 mg l−1 ) of this salt was prepared daily by dissolving 10 mg of it in 100 ml of bi-distilled water with the use of ultrasonic bath. All other reagents used were of analytical grade and were obtained from POCh-Gliwice, Poland or Aldrich (NBS). The solution of NBS (0.04 mol l−1 ) was prepared daily by dissolving 3.558 g of this substance in 0.5 l of bi-distilled water. Phenols and organic amines solutions were prepared daily, but stock solutions (1000 mg l−1 ) of glycerine, rhodamine B, rhodamine 6G, dichlorofluorescein and calcein were stable for few weeks at room temperature. 2.2. Apparatus The flow injection set-up, shown schematically in Fig. 1, consisted of an Ismatec MS-Reglo peristaltic pump, a Model 5021 rotary injection valve (Rheodyne, Cotati, CA), and flow luminometer (KSP, Poland) with
3. Results and discussion 3.1. Optimization of parameters 3.1.1. Concentration of sodium hydroxide The influence of the concentration of sodium hydroxide in a carrier solution was investigated in the range of 0.25–3 mol l−1 . The maximum response was observed for 2 mol l−1 solution of sodium hydroxide and this concentration was chosen as the optimal one (Fig. 2A). The reproducibility was also worse at the concentration of 3 mol l−1 , which is connected with the increased viscosity of the carrier stream.
Fig. 1. Flow injection manifold used for determination of humic acid in natural waters: C: 2 mol l−1 sodium hydroxide solution, R: 0.04 mol l−1 NBS solution, P: peristaltic pump, S: injection valve, F: flow cell, L: luminometer, PC: personal computer, W: waste; distance between merging point and flow-cell was 10 cm.
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Fig. 2. Optimization of the parameters: A: sodium hydroxide concentration, B: NBS concentration, C: pumping velocity, D: sample volume.
3.1.2. Concentration of NBS Concentration of NBS in the reagent solution is an important factor influencing signal magnitude. The increase of the signal was observed until 0.05 mol l−1 (Fig. 2B), which is the limit of solubility at room temperature. At this concentration, poor reproducibility was the limiting factor and the concentration of 0.04 mol l−1 was used in further experiments. Other reason for choosing the concentration of 0.04 mol l−1 was saving of the reagent. 3.1.3. Pumping velocity The chemiluminescence was found to increase constantly with increasing pumping velocity, due to high speed of the chemiluminescent reaction. The pumping speed higher than 5.9 ml min−1 caused poor reproducibility, so this flow rate was chosen for both streams (Fig. 2C). At a high pumping velocity the flow becomes more turbulent and irreproducible, so our choice was the compromise between the peak height and reproducibility. 3.1.4. Sample volume An increase of the signal magnitude while increasing the sample volume was observed (Fig. 2D). By the sample volume higher than 400 l double peaks were recorded, so this volume was considered to be
optimal one. Splitting of the peaks was caused by the differences in the reaction velocity in the middle of the sample zone and at its edges, which often happens when the sample volume is too high. 3.1.5. Temperature Increasing the temperature of the carrier and reagent solutions does not influence the signal magnitude in a considerable degree. The slope of the calibration graph was very low at the temperature 30◦ C and higher, so the room temperature (18–20◦ C) was considered as optimal for the determination. 3.2. Parameters of the optimized method A calibration graph made with the use of commercial available humic acid salt was linear in a wide concentration range. Two working ranges of calibration were established: 0.012–1 and 1–2 mg l−1 . All calibration parameters of the optimized system are collected in the Table 1. The maximal sample throughput in the optimized system was 180 h−1 . 3.3. Effect of sensitisers An addition of different fluorofores like rhodamineB, rhodamine-6G, 3,5-dichlorofluorescein or calcein
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Table 1 Calibration parameters of the optimised system depicted in Fig. 1 Parameter
Value
Calibration range
0.012–1.0 mg l−1 , y = 0.017x + 0.6907, r 2 = 0.9909 1.0–2.0 mg l−1 , y = 17.714x + 4.2276, r 2 = 0.9953 0.012 mg l−1 3.2% (0.02 mg l−1 ) 1.2% (1.0 mg l−1 )
Limit of detection R.S.D. (n = 15)
to the sample resulted in no signal enhancement. Compounds used as sensitisers in other procedures utilizing NBS, like ammonium [17–19] or hydroxylammonium hydrochloride [23] do not enhance chemiliminescence resulting from the oxidation of humic acid. Great signal enhancement (about 100-fold) was achieved after the addition of glycine (aminoacetic acid) to the sample. The signal obtained for the mixture of humic acid and glycine is considerably higher than the sum of signals for both compounds separately. The optimal concentration of the sensitise was found 35 mg l−1 . A further increase of the concentration of glycine in the sample resulted in no signal enhancement. 3.3.1. Mechanism of the signal enhancement Among many amino compounds like ammonium, hydrazine, hydroksylamine hydrochloride, ethanolamine, n-buthylamine and glycine, only the last one produces signal enhancement in the oxidation reaction of humic acid. Because it is unlikely that glycine acts as simple fluorophore, it was supposed that it reacts with some functional group(s) of humic acid and product(s) of this reaction give rise to the signal magnitude. The most important, from the analytical point of view, functional groups of humic acid are: phenolic hydroxyl, carboxylic, carbonyl and quinone groups [24]. Many model compounds with functional groups present in humic acid particles were tested for chemiluminescent reaction with glycine. Signals of analytical importance (S/N > 3) were observed for 10 mg l−1 solutions of phenol, o-nitrophenol, resorcinol, hydroquinone, pyrogallol, but without its enhancement after the addition of glycine at 35 mg l−1 . No signals were observed for 10 mg l−1 solutions of benzoic and salicylic acids and for benzoquinone solution of the same concentration. After the addition of glycine to them, only small signal characteristic of
this substance was detected. The conclusion is that the sensitising effect cannot be attributed to the presence of phenolic, quinone or carboxylic functional groups in humic acid particles. In the same way, groups containing sulphur, like in thiophenol, 1,2-ethanothiol and thiourea were tested with negative results. Aromatic carbonyl groups were found to react with glycine, and product(s) of this condensation reaction is supposed to affect the mechanism of chemiluminescence production. An addition of glycine at concentration of 35 mg l−1 to the solution of salicylic aldehyde, benzaldehyde, p-diethylaminobenzaldehyde or p-phtalaldehyde (10 mg l−1 ) gives two to five-fold signal enhancement compared with the sum of the signals for glycine and aldehyde in separated solutions. The highest signal enhancement, five-times, was achieved for salicylic aldehyde. A reaction between amino acids and o-phtalaldehyde in the alkaline medium was used for derivation of them, which enables its sensitive fluorimetric determination [25]. Strong fluorescence of the product of this reaction suggests that its chemiluminescent activity is possible. 3.4. Study of interferences Organic and inorganic compounds typically occurring in natural waters were studied as potential interferents. Each substance was considered not to interfere if it caused a relative error less than 5% for the determination of 0.5 mg l−1 of humic acid. The results are shown in Table 2. Most of the compounds at the concentration typical of natural waters do not interfere by the determination. Only phenols with one hydroxyl group should be considered as serious interferents, which affect the signal magnitude in the concentration higher than 0.075 mg l−1 . Because these compounds are volatile, they could be removed by evaporation.
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Table 2 Study of interferences with the determination of 0.5 mg l−1 of humic acid in the proposed method Interferent
Maximum tolerated concentration (mg l−1 )
Relative error (%)
Na+
1000.0 800.0 80.0 80.0 8.0 5.0 2.0 150.0 5.0 0.5 20.0 200.0 500.0 1000.0 200.0 0.5 800.0 1000.0 0.5 5.0 1.0 5.0 5.0 0.2 5.0 2.0 15.0 20.0 0.075 0.05 0.05
−4.9 −2.4 −1.3 2.2 −1.7 −4.9 −3.5 −2.7 −2.2 −0.8 2.3 −3.5 4.9 0.2 3.9 1.3 1.7 −4.9 −0.2 3.4 −4.7 −0.6 −4.4 −4.9 −1.2 −4.3 −1.0 −2.3 −0.2 −4.7 −5.0
K+ NH4+ Ca2+ Mg2+ Fe2+ Fe3+ Zn2+ Cu2+ Cr3+ CrO4 2− Cl− CO3 2− SO4 2− SO3 2− S2− PO4 3− NO3 − NO2 − Hydroxylamine Hydrazine 2,4-Diphenylhydrazine Diphenylamine p-Nitroaniline Pyrogallol Pyrocatechol Resorcinol Hydroquinone Phenol p-Cresol 2,4,6-Trichlorophenol
For this purpose 100 ml of the sample was gently boiled until its volume was reduced to about 60 ml. This solution was transferred after cooling to the 100 ml volumetric flask, appropriate volume of the sensitise was added, and it was adjusted to the mark with bi-distilled water. The validity of this procedure of interference removal was established for solutions containing 1 mg l−1 of humic acid and 1 mg l−1 of phenol, p-cresol or phenol + p-cresol. In each case a relative error of determination was less than 1%. 3.5. Analysis of real samples In order to perform determination of humic acid in real samples standard of this substance was
prepared according to Thurman and Malcolm [26]. For this purpose, humic acid was extracted from the river water of Czarna river, which is located in Bialystok’s forest area (15 km from Bialystok). The determination of humic acid using obtained standard was performed with both, proposed and spectrophotometric [27] methods in the water samples collected from the same river. The results of determination are shown in the Table 3. Parameters of the calibration with the prepared standard were very similar to those for humic acid purchased by Aldrich: equation y = 16.157x + 2.337 for the calibration graph in the range 1–2 mg l−1 , r 2 = 0.9965, R.S.D. (n = 15) 3.4 % at the concentration 0.02 mg l−1 and the detection limit 0.014 mg l−1 .
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Table 3 Comparison of the results of determination of humic acid in real samples with the proposed and spectrophotometric methods; humic acid standard was prepared according to [26]a Sample
River 1 River 2 River 3 a
Concentration of humic acid (mg l−1 ) Proposed method (main value ± S.D.)
Spectrophotometric method [27] (main value ± S.D.) (%)
Relative error (%)
6.4 ± 0.2 5.9 ± 0.1 5.7 ± 0.1
6.6 ± 0.3–3.0 5.8 ± 0.3 5.8 ± 0.2
−3.0 1.7 −1.7
Humic acid was extracted from the river water of Czarna river.
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