J S S
ISSN 1615-9306 · JSSCCJ 38 (5) 703–882 (2015) · Vol. 38 · No. 5 · March 2015 · D 10609
Methods Chromatography · Electroseparation Applications Biomedicine · Foods · Environment
852 Tha´ıs Tamye Marques1 Let´ıcia Sayuri Shiroma1 Dosil Pereira de Jesus1,2 1 Institute
of Chemistry, University of Campinas, UNICAMP, Campinas, SP, Brazil 2 Instituto Nacional de Ciencia ˆ e Tecnologia de Bioanal´ıtica, Campinas, SP, Brazil Received November 15, 2014 Revised December 15, 2014 Accepted December 17, 2014
J. Sep. Sci. 2015, 38, 852–857
Determination of tetrakis(hydroxymethyl)phosphonium sulfate in commercial formulations and cooling water by capillary electrophoresis with contactless conductivity detection A novel capillary electrophoresis method using capacitively coupled contactless conductivity detection is proposed for the determination of the biocide tetrakis(hydroxymethyl)phosphonium sulfate. The feasibility of the electrophoretic separation of this biocide was attributed to the formation of an anionic complex between the biocide and borate ions in the background electrolyte. Evidence of this complex formation was provided by 11 B NMR spectroscopy. A linear relationship (R2 = 0.9990) between the peak area of the complex and the biocide concentration (50–900 mol/L) was found. The limit of detection and limit of quantification were 15.0 and 50.1 mol/L, respectively. The proposed method was applied to the determination of tetrakis(hydroxymethyl)phosphonium sulfate in commercial formulations, and the results were in good agreement with those obtained by the standard iodometric titration method. The method was also evaluated for the analysis of tap water and cooling water samples treated with the biocide. The results of the recovery tests at three concentration levels (300, 400, and 600 mol/L) varied from 75 to 99%, with a relative standard deviation no higher than 9%. Keywords: Biofilms / Biofouling / Cooling water / Sulfate-reducing bacteria / Water treatment DOI 10.1002/jssc.201401288
1 Introduction Biocides are compounds that are widely used for controlling the growth of microorganisms such as bacteria, fungi, and algae in water used in industrial processes, particularly in cooling systems. This water treatment avoids slime and biofilm formation in the inner parts of equipment, the formation of which can cause major economic and technical problems. Depending on the chemical properties and the mechanism used to kill the microorganisms, biocides are usually classified as oxidants or nonoxidants . For instance, chlorine, bromine, hydrogen peroxide, and ozone are oxidant biocides, whereas quaternary ammonium salts, carbamates, glutaraldehyde, organothiocyanates, biguanides, isothiazolins,
Correspondence: Dr. Dosil Pereira de Jesus, Institute of Chemistry, University of Campinas, P.O. Box 6154, 13083-970 Campinas, SP, Brazil Fax: +5519-3521-3023 E-mail: [email protected]
Abbreviations: C4 D, capacitively coupled contactless conductivity detection; MES, 2-(N-morpholino)ethanesulfonic acid; SRB, sulfate-reducing bacteria; THP, trihydroxymethylphosphine; THPO, trishydroxymethylphosphine oxide; THPS, tetrakis(hydroxymethyl)phosphonium sulfate C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
tetrakis(hydroxymethyl)phosphonium sulfate (THPS), and 2,2-dibromo-3-nitrilo-propionamide are nonoxidant biocides. THPS is a fully water-soluble quaternary phosphonium salt (Fig. 1) that shows antibacterial properties, especially against sulfate-reducing bacteria (SRB) . This feature makes THPS suitable not only for traditional water treatment but also in oilfield applications to avoid corrosion and clogging of metallic ducts by iron sulfides due to the formation of H2 S by SRB activity [2, 3]. In aqueous solutions, THPS slightly dissociates (Fig. 2A) to form trihydroxymethylphosphine (THP) [1, 4, 5]. The biocide activity attributed to THPS is in fact due to THP, which has the ability to inactivate the proteins in the cell walls of microorganisms by reducing the disulfide bonds of cystine residues, thus converting them to cysteine groups . THPS is considered an environmentally friendly biocide because it is easily degraded by oxidation to trishydroxymethylphosphine oxide (THPO). In aquatic environments, THPO is considered to be a very low toxicity agent that can be further mineralized to form carbon dioxide, water, and phosphate . Determination of the active concentration of THPS in water is important for the control and establishment of effective water treatment, for monitoring industrial effluents, and for degradation studies . Nevertheless, analytical methods for the determination of THPS are rarely described in the www.jss-journal.com
J. Sep. Sci. 2015, 38, 852–857
were analyzed. To the best of our knowledge, this is the first time CE was used for THPS determination.
2 Materials and methods 2.1 Reagents, samples, and solutions Figure 1. Structure of THPS.
literature . The classic starch-iodine titration has been used as the standard method for the determination of THPS in water and commercial formulations [7,8]. However, this method involves a large amount of sample and reagents, and it is subject to interference from other compounds in the sample. A more accurate method involves ion chromatography separation with a postcolumn reaction for spectrophotometric detection . CE has the potential to provide measurements of biocide concentrations in a short time and with low consumption of samples and reagents . However, this CE application has only been reported by a few authors [10–13]. CE using capacitively coupled contactless conductivity detection (C4 D) [14,15] is a suitable approach for the detection of non-UV-absorbing compounds, and it has been used for the determination of several inorganic and organic species in many matrices [16–19]. In this paper, a novel and simple CE–C4 D method is described and evaluated as an alternative analytical method for the determination of THPS in water. Using this method, commercial formulations of THPS and four different samples of tap water and cooling water treated with the biocide
All reagents were of analytical grade, except for the THPS aqueous stock solution (70–75%) that was purchased from Sigma–Aldrich (Saint Louis, MO). This stock solution was standardized by an iodometric titration method (described later), and the concentration of THPS was determined to be (80.5 ± 0.5)% w/w. Sodium tetraborate decahydrate (borax), potassium iodide, iodine, dehydrate sodium dichromate, and sodium thiosulfate pentahydrate were purchased from Synth (Diadema, Brazil), and 2-(N-morpholino)ethanesulfonic acid monohydrate (MES) was obtained from J. T. Baker (Philipsburg, NJ). Ultrapure water was obtained from a Direct-Q3 UV Water Purification System (Millipore, Molsheim, France). The BGE was a 20 mmol/L borate buffer aqueous solution (pH 9.2). Standard solutions of THPS were prepared by dilution of the stock solution with the BGE. MES solution was added (500 mol/L) to all solutions as an internal standard. The commercial formulations of THPS containing 75 and 50% w/w biocide were provided by the Rhodia Solvay Group (S˜ao Paulo, Brazil). Tap water samples were collected from our laboratory, and samples of water used in circulating cooling systems were obtained at the Institute of Chemistry of the University of Campinas (IQ-Unicamp) and Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, Brazil.
Figure 2. (A) Dissociation reaction of THPS in water and (B) proposed reaction between borate ion and the THP to form an anionic complex THP-borate.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Sep. Sci. 2015, 38, 852–857
T. T. Marques et al.
2.2 Instrumentation and procedures 2.2.1 CE analyses The CE analyses were conducted in a homemade CE system equipped with an in-house made C4 D . A software implemented in Labview 8.2 (National Instruments, Austin, TX, USA) was used to control the CE system and to acquire the C4 D signal at a sampling frequency of 3.3 Hz. A bore fused-silica capillary column 40.0 cm in length (32.0 cm effective) with a 50 m id was used for the CE separations. The standardized and sample solutions were hydrodynamically injected into the capillary column by pressure (11 kPa) for 5 s. The CE separation, in the counter EOF mode, was conducted with a potential of 25 kV and the C4 D operating at 620 kHz (sinusoidal) and 1.5 V (peak to peak amplitude). All operations were carried out at ambient temperature (20– 25⬚C). Before the first analysis of the day, the fused-silica capillary was sequentially washed with 0.1 mol/L NaOH, water, and BGE (5 min each). After each run, the capillary was flushed with BGE for 30 s. Analytical curves were obtained by injecting (in triplicate) six standard solutions of THPS at concentration levels from 50 to 900 mol/L. The areas of the peaks in the electropherograms were integrated, and the analytical curves were then plotted as a ratio of the area of the peak related to the analyte to that of the internal standard versus the THPS concentration. A linear regression was performed on the standard curves using the least-square method, and the obtained regression equations were used to estimate the concentrations of the analytes in the samples. The peak integration and the statistical analysis were carried out with Origin 8.1 software (OriginLab, Northhampton, MA, USA).
Figure 3. Electropherograms obtained from a cooling water sample (A) without and (B) with the addition of THPS (300 mol/L), and from a (C) standard solution of THPS (500 mol/L). BGE: 20 mmol/L sodium borate solution (pH 9.2). Fused-silica capillary column 40 cm in length (32 cm effective) with a 50 m id. Separation voltage of 25 kV; pressure injection at 11 kPa for 5 s; C4 D detection at 620 kHz (sinusoidal) and 1.5 V (peak to peak amplitude). Peaks: (*) unidentified, (1) THP-boron complex, (2) MES (internal standard).
internal standard (MES) was added (500 mol/L) to all samples before the CE separations.
2.4 Investigation of THPS complex formation by NMR spectroscopy 11
2.2.2 Standard iodometric titration method An analytical-grade THPS reagent was not available for purchase. Instead, a commercial aqueous THPS solution (70– 75% w/w) was standardized (n = 5) by the iodometric titration method  using a 0.1 mol/L iodine solution previously standardized with a sodium thiosulfate solution (0.1 mol/L). This method was also used to evaluate the accuracy of the CE–C4 D method for the analysis of the samples of commercial THPS formulations.
2.3 Sample preparation The analyzed commercial formulations of THPS were only diluted with the BGE before injection into the CE–C4 D system. The samples of water were treated with three different THPS concentration levels (300, 400, and 600 mol/L). This concentration range is generally used in the treatment and control of micro-organisms in water. After the THPS addition, the samples were diluted (1:1 v/v) with the BGE. The C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
B NMR spectra were obtained using a Bruker Avance III 500 spectrometer operating at 160 MHz. The chemical shifts were measured from BF3 ·Et2 O, used as an external reference (0 ppm). Three aqueous solutions containing the same concentration of borate (50 mmol/L) and different THPS concentrations (0, 25, and 50 mmol/L) were analyzed.
3 Results and discussion 3.1 CE separation Figure 3 shows electropherograms from a standard solution of THPS and from a sample of cooling water before and after the addition of THPS. The negative peaks obtained with C4 D means that the detected species have conductivities (or electrophoretic mobilities) lower than the BGE. Peak 1 corresponds to an anionic species, and its peak area was demonstrated to have a linear relationship with the THPS concentration. THPS barely dissociates (pKd = 12.20) in water to form tris(hydroxymethyl)phosphine (THP), formaldehyde, and sulfuric acid (Fig. 2A) . However, THPS and THP www.jss-journal.com
J. Sep. Sci. 2015, 38, 852–857
Table 1. Figures of merit for the CE–C4 D method
Migration time (min)a) N/mb) Rs c) Regression equationd) LOD (mol/L)e) LOQ (mol/L)f) Linear range (mol/L) Determination coefficient (R2 ) Intraday instrumental precision (%)g)
Figure 4. 11 B NMR spectra of a sodium borate solution (50 mmol/L, pH 9.2) containing (A) 0, (B) 25, and (C) 50 mmol/L of THPS.
are neutral compounds and could not be separated by CE. Nevertheless, it was found that in borate buffer solutions, the equilibrium of the reaction shown in Fig. 2A was strongly displaced toward the products. This increase in dissociation was confirmed by the determination of the sulfate concentration (data not shown) using the same CE–C4 D method. As will be discussed in the next section, peak 1 (Fig. 3) can be ascribed to a borate complex with THP (Fig. 2B). By contrast, the area of the unidentified peak did not vary with the THPS concentration and can most likely be attributed to some degradation product of the biocide. MES was demonstrated to be a suitable internal standard because it was not found in detectable amounts in the analyzed samples and its peak showed good resolution as well as a migration time close to that of the analyte.
3.2 THP-boron complex formation The existence of an anionic borate complex with THP was investigated by 11 B NMR spectroscopy. Figure 4 shows the 11 B NMR spectra of a sodium borate solution (50 mmol/L) before and after addition of THPS at two different concentrations. The NMR spectrum of the solution containing only borate buffer (Fig. 4A) showed a broad peak with a chemical shift of 10.6 ppm. However, a sharper peak was observed at 19.5 ppm when THPS was present (Fig. 4B and C). The intensity of this peak increased with the THPS concentration, suggesting an interaction between the borate and the biocide. It is well known that boric acid or borate ions can react with compounds having various hydroxyl groups (polyols) to form anionic complexes . Moreover, the formation of a borate complex with compounds containing hydroxymethyl groups, such as tris(hydroxy-methyl)aminomethane (Tris), has also been reported . THPS and THP have hydroxymethyl groups that could form a complex with borate ions. Because THP is the major species in the borate buffer (Fig. 2A), it was proposed that THP reacted with the borate C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1.16 ± 0.02 14 686 1.4 y = 0.0024x – 0.019 15.0 50.1 50–900 0.9990 3.6
a) Mean and standard deviation for 15 consecutive injections. b) Number of plates (N) per meter; N = 5.54 (tm /w1/2 )2 , where tm = migration time and w1/2 = peak width at half height. c) Resolution between the unidentified peak and THP-complex peak; Rs = 2(tm2 – tm1 )/(w1 + w2 ), where tm1 and tm2 are the migration times and w1 and w2 are the widths at peak base. d) x = concentration of THPS (mol/L); y = ratio of the peak area of THP-complex to that of the internal standard. e) S/N = 3. f) S/N = 10. g) RSD for the ratio of the peak area of THP-complex to that of the internal standard for consecutive injections (n = 15) of a 500 mol/L THPS standard solution.
ions as depicted in the reaction shown in Fig. 2B. According to this proposed reaction, an anionic THP-borate complex was formed when the biocide standard solutions, or the treated water samples, were diluted with the BGE containing borate ions. By using the CE–C4 D method, the anionic complex was then separated and detected (Fig. 3) for measurement of the concentration of THPS. According to previous studies , the electrophoretic mobility of borate complexes with charge-neutral compounds is influenced by the concentration of borate and pH of the BGE. Nevertheless, an optimization of these parameters was not required because the used BGE (20 mmol/L borate solution, pH 9.2) provided an efficient separation and a good compromise between buffer capacity and low Joule heating. Under these separation conditions the electrophoretic mobility of the THP-borate complex was calculated to be –2.23 × 10–4 cm2 /Vs.
3.3 Figures of merit of the CE–C4 D method The main figures of merit for the CE–C4 D method were obtained (Table 1) according to the recommendations of the literature for the validation of analytical separation methods . A CE separation run required less than 2 min, and the RSD for the migration time of the analyte was 1.6%. The LOQ was lower than the recommended concentrations (175–650 mol/L) for THPS in the treatment of cooling water (www.dowmicrobialcontrol.com). For the evaluated concentration range (50–900 mol/L), the CE–C4 D method showed a good determination coefficient (R2 = 0.9990) and linearity that was validated by the lack of fit test . The repeatability www.jss-journal.com
J. Sep. Sci. 2015, 38, 852–857
T. T. Marques et al.
Table 2. Analysis results for the determination of THPS in commercial formulations
Labeled concentration (%)a)
Concentration measured (%)b) (mean ± SD)b)
48 ± 2 79 ± 4
47.9 ± 0.4 77 ± 1
a) (weight/weight). b) n = 3 and 5 for the CE-C4 D and titration method, respectively. c) Calculated Student’s t-value; 2.54 is the tabulated t-value at a confidence level of 95% and 6 degrees of freedom. Table 3. Average recoveries (%) ± RSDa) at three concentration levels of THPS
Concentration added (mol/L)
300 400 600
Samplesb) Tap water
93 ± 7 94 ± 4 97 ± 6
98 ± 4 99 ± 9 99 ± 3
75 ± 7 90 ± 5 86 ± 7
85 ± 5 89 ± 5 91 ± 6
Figure 5. Electropherograms of a cooling water sample (A) as soon as the treatment with THPS (300 mol/L) was performed and (B) three days after the treatment. Separation conditions and peak attribution as in Fig. 3.
The recoveries ranged from 75 to 99% with RSD no higher than 9%. These results can be considered acceptable for the validation of analytical separation methods .
a) n = 3. b) CW = cooling water.
3.6 Degradation assay for THPS of the peak area was 3.6% for consecutive injections (n = 15) of a standard THPS solution (500 mol/L). 3.4 Analysis of commercial THPS formulations Table 2 displays the results of the analysis of two commercial formulations of THPS by the CE–C4 D method. The standard iodometric titration method was also conducted for comparison purposes. The concentration values obtained by both methods were considered statistically similar by Student’s ttest at a confidence level of 95%. The concentrations were also close to those reported by the manufacturer. Nevertheless, the CE–C4 D method required lower amounts of sample and reagents (hundreds of microliters) and generated a smaller amount of residues compared to the titrimetric method. These results suggest that the proposed method is a good alternative for application in quality control of commercial formulations of THPS. 3.5 Analysis of water samples The available amount of some water samples was not sufficient to conduct the iodometric titration analysis. Instead, the accuracy of the CE–C4 D method for the determination of THPS in samples of tap water and cooling water was evaluated by recovery tests (Table 3) at three concentration levels (low, medium, and high). C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The CE–C4 D method was also evaluated for the detection of THPS degradation. Figure 5A displays the electropherogram of a sample of cooling water as soon as the sample was treated with THPS. This same sample was again injected in the CE–C4 D system three days after the treatment (Fig. 5B). An unidentified peak appeared then, and the peak area of the THP-borate complex decreased, suggesting degradation of the biocide. These results demonstrated that the proposed method has the potential to be used in studies of the degradation of the THPS, particularly in water.
4 Concluding remarks The anionic species formed by the interaction between the borate ions in the BGE with the THP allowed the determination of the biocide THPS by CE–C4 D. The proposed method was demonstrated to be rapid, simple, precise, and accurate for monitoring THPS in water, particularly treated water used in cooling systems. Compared to the standard iodometric titration method, the CE–C4 D method has the advantage of requiring a lower amount of sample and reagents, and a smaller amount of residues was generated. The proposed method also demonstrated potential for monitoring THPS degradation in water. This work was supported by the Fundac¸a˜o de Amparo a` Pesquisa do Estado de S˜ao Paulo (FAFESP, grants www.jss-journal.com
J. Sep. Sci. 2015, 38, 852–857
no . 2013/22485-6) and the Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico (CNPq, grants no . 305318/20128). The authors thank Jos´e Alberto Fracassi da Silva for providing the CE-C4 D equipment, and Leandro Y. Shiroma and the Centro Nacional de Pesquisa em Energia e Materiais (CNPEM) for providing the samples of cooling water. The authors are grateful to ´ Eder Rissi Torres and the Rhodia Solvay Group for the donation of the THPS sample formulations. The authors declare no conflicts of interest.
5 References  Paulus, W., Microbicides for the Protection of Materials—A Handbook, Chapman & Hall, London 1993.  Gana, M. L., Kebbouche-Gana, S., Touzi, A., Zorgani, M. A., Pauss, A., Lounici, H., Mameri, N., J. Ind. Microbiol. Biot. 2011, 38, 391–404.  Xu, D., Li, Y., Gu, T., World J. Microb. Biot. 2012, 28, 3067–3074.
 Albright & Wilson Water Management Chemicals Technical Bulletin No 230, “Analytical Methods for the Determination of THPS”.  Kemp, G., Biotechnol. Appl. Bioc. 1998, 27, 9–17.  Abrantes, S., Philo, M., Damant, A. P., Castle, L., J. Microcolumn Sep. 1998, 10, 387–391.  Fekete, A., Hertkorn, N., Frommberger, M., Lahaniatis, M. R., Kettrup, A., Schmitt-Kopplin, P., Electrophoresis 2006, 27, 2216–2224.  Jones, T. L., Riddick, L., J. Capillary Electrop. 1997, 4, 33–37.  Para, B. V., Nunez, O., Moyano, E., Galceran, M. T., Electrophoresis 2006, 27, 2225–2232.  da Silva, J. A. F., do Lago, C. L., Anal. Chem. 1998, 70, 4339–4343.  Zemann, A. J., Schnell, E., Volgger, D., Bonn, G. K., Anal. Chem. 1998, 70, 563–567.  Kuban, P., Hauser, P. C., Electrophoresis 2009, 30, 176– 188.  Kuban, P., Hauser, P. C., Electrophoresis 2013, 34, 55–69.  Ji, Y. L., Chen, X. W., Zhang, Z. B., Li, J., Xie, T. Y., J. Sep. Sci. 2014, 37, 3000–3006.
 Hu, T. Q., Williams, T., Schmidt, J. A., James, B. R., Cavasin, R., Lewing, D., Pulp Pap-Canada 2009, 110, 37– 42.
 da Silva, I. S., Vidal, D. T. R., do Lago, C. L., Angnes, L., J. Sep. Sci. 2013, 36, 1405–1409.
 Frank, A. W., Daigle, D. J., Vail, S. L., Text. Res. J. 1982, 52, 678–693.
 Tuma, P., Malkova, K., Wedellova, Z., Samcova, E., Stulik, K., Electrophoresis 2010, 31, 2037–2043.
 Lacorte, S., Latorre, A., Barcelo, D., Rigol, A., Malmiqvist, A., Welander, T., TrAC, Trends Anal. Chem. 2003, 22, 725– 737.
 Tournie, A., Majerus, O., Lefevre, G., Rager, M. N., Walme, S., Caurant, D., Barboux, P., J. Colloid Interf. Sci. 2013, 400, 161–167.
 Van Esch, G. J., International Programme on Chemical Safety, The United Nations Environment Programme, International Labour Organisation, World Health Organization, Bilthoven, the Netherlands 2000.
 Ribani, M., Bottoli, C. B. G., Collins, C. H., Jardim, I. C. S. F., Melo, L. F. C., Quim. Nova 2004, 27, 771–780.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
 Danzer, K., Currie, L. A., Chem, C. G. A. A., Pure Appl. Chem. 1998, 70, 993–1014.