In the Laboratory
Capillary Electrophoretic Quantitative Analysis of Anions in Drinking Water Stéphane Demay, Alexia Martin-Girardeau,* and Marie-France Gonnord Département de Chimie, Laboratoire des Mécanismes Réactionnels, URA UMR 7651, Ecole Polytechnique, F91128 Palaiseau, France; *
[email protected] Nowadays the public is increasingly aware of environmental pollution, because it is well known that numerous health problems are linked to it. To address these issues, drastic standards have been imposed on industry. At the same time, new analytical techniques have been devised to detect and quantify a large range of pollutants that may be present in very small quantities. Water and air have been particularly closely studied and monitored because they are the most efficient pollution carriers, and human activities are directly responsible for their contaminants. A great number of substances are present in water: for example, nitrate ion, which shows toxicity in humans if its concentration is greater than 50 mg/L. High nitrate concentrations often result from the intensive use of natural or chemical fertilizers. High-performance liquid chromatography (HPLC) is the most widely used analytical tool for determining the concentration of anions (chlorides, sulfates, carbonates, nitrites, nitrates, bromides, or phosphates) in drinking water. We developed a procedure based on capillary electrophoresis (CE) to rapidly and effectively analyze anions present in river water and noncarbonated mineral water. Several published articles inspired this work (1–4). Our procedure has been tested successfully several times with groups of students as part of their laboratory courses. In this fashion, we aimed at demonstrating the rapidity and ease of CE in comparison with traditional methods like HPLC. Furthermore, students are introduced to the practice and theory of CE, a relatively new analytical technique. This provides an environmentally relevant laboratory experience for undergraduate students in analytical chemistry or environmental chemistry. Principles and Applications of the Method CE is a method of separation based on the differential migration of charged species in solution under the influence of an applied electric field. Although the principle of electrophoresis has been known for a long time, it was not until 1937 that Tiselius demonstrated the separation of natural amino
Term Symbol Units ––––––––––––––––––––––––––––––––––––––––––––––––––––– electrophoretic mobility µ ep cm 2 V {1 s {1 electrophoretic velocity Vep cm s {1 electric field E V cm {1 viscosity η Pa s ion radius r cm ion charge q C permittivity of free space εo 8.854 × 10{12 F m {1 relative dielectric constant εr C 2 J{1 m{1 zeta potential ζ V
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acids. Jorgenson and colleagues in the early 1980s clearly delineated rules for capillary electrophoresis and extended CE to a broad range of applications. Thus it is possible to establish a clear relationship between operational parameters and the quality of the separation. Highly sophisticated equipment, allowing real-time data processing and automation, has appeared on the market. The technique is undergoing rapid development, and can be applied to an increasing variety of solutes and separation problems. Basic principles of CE are quite simple. The symbols for the terms used in this discussion and the units in which they are expressed are given in the box. As shown in Figure 1, an open tubular fused-silica capillary is filled with a buffer electrolyte. Sample introduction is accomplished by either hydrodynamic (pressure, vacuum, or gravity) or electrokinetic injection. An electric field is then applied between two buffer reservoirs, into which the ends of the capillary are immersed. The charged species migrate to the corresponding electrodes. They move according to two physical phenomena: the electrophoretic mobility (µep) and the electroosmotic flow (EOF). The resulting mobility is called apparent mobility (µapp).
Electrophoretic Mobility The electrophoretic mobility µep is defined by the ratio of migration velocity (Vep) of an ion with the applied value of the electric field (E ) µep = V ep/E
(1)
where µep can be either positive or negative, depending on the sign of the ions’ charge. For neutral species, µep is zero. The limiting velocity is reached when the electric (Fe ) and the frictional (Ff) forces are equal but opposite. Fe = qE
(2)
Ff = 6 πη rVep
(3)
where η is viscosity, r is ion radius, and q is ion charge. Then, from eqs 1–3, µep= q/(6πη r)
From this equation, it is evident that small, highly charged species have high mobilities, whereas large, minimally charged species have low mobilities.
Electroosmotic Flow The electroosmotic flow is the consequence of the buffer displacement throughout the capillary wall resulting from the effect of the applied electric field. This phenomenon can be briefly explained in the following manner. The fused silica capillaries typically used for separation bear ionizable silanol groups (SiOH) (pI = 1.5) that deprotonate as soon as the pH is above 2 and form a polyanionic layer of SiO{. The negatively charged wall attracts positively charged ions from the buffer, creating an electrical double layer (ζ potential). When a voltage
Journal of Chemical Education • Vol. 76 No. 6 June 1999 • JChemEd.chem.wisc.edu
In the Laboratory
Apparent Mobility The apparent mobility is the sum of the electrophoretic mobility and the electroosmotic flow (Fig. 2). If the capillary wall is uncoated, the apparent mobility of a cation is greater than the apparent mobility of an anion with the same massto-charge ratio. Therefore they are first detected at the cathode.
Figure 1. Basic configuration of a capillary electrophoresis system.
Figure 2. Development of the apparent velocity.
Figure 3. Elimination of the electroosmotic flow using a cationic surfactant.
Analysis of Anions To separate and quantify anions, operative parameters need to be optimized, thus modifying typical analytical conditions of capillary electrophoresis. First the polarity of the electrodes can be reversed so that the UV detector is 7 cm from the anode, as hydrodynamic injection occurs at the cathode. Then the electroosmotic flow is reduced. The run is then operated in the so-called reversed-polarity manner. EOF Control Several experimental parameters can be used to modify and control the EOF: electric field, pH, buffer ionic strength, or temperature. For this application, most of these parameters present disadvantages: either they raise the time of analysis or they lessen the resolution and efficiency of the separation. For this reason, a cationic surfactant (quaternary ammonium type) is introduced into the buffer to decrease EOF by coating the capillary wall and neutralizing the charge of the deprotonated silanol groups (Fig. 3). However, the surfactant concentration cannot be allowed to be too large; otherwise the EOF is reversed rather than decreased by creating a second layer of surfactant that results from Van der Waals interactions between the hydrophobic tails (Fig. 4). In such a case, the separation deteriorates because anions migrate too rapidly. Detection The detection of anions is accomplished by indirect UV absorbance because most of the anions do not show sufficient UV absorptivity. A chromophore that absorbs in the UV range is added to the buffer, and displacement by the analyte anions permits indirect photometric detection. Sodium chromate is used for its electrophoretic mobility close to that of the anion and for its capability of giving negative, symmetrical peaks. The software permits inversion of the signal in order to integrate peak areas in a conventional manner. Experimental Procedure
Figure 4. Reversal of the electroosmotic flow using a cationic surfactant.
is applied across the capillary, cations in the diffuse portion of the double layer migrate in the direction of the cathode, carrying a bulk flow of buffer toward the cathode. The magnitude of the EOF can be expressed in terms of velocity by the relation µEOF = V EOF /E = ( { ε oε r ζ /η )
where VEOF is velocity of EOF, ε o is permittivity of free space, r is relative dielectric constant, ζ is zeta potential, and η is viscosity.
The experimental procedure developed for this laboratory training course has been applied to the detection of chlorides, sulfates, fluorides, and nitrates in the concentration range 2–100 ppm. These concentrations are in an appropriate range for the analysis of drinking water, mineral waters, and river water.
Equipment and Material Capillary electrophoresis was performed using a P-ACE 2100 (Beckman, Palo Alto, CA). Data were collected and analyzed with the Beckman System Gold Software. Uncoated fused silica capillary tubing with an internal diameter of 75 µm (a total length of 47 cm and a length from inlet to the detector of 40 cm) was purchased from Beckman and installed in a cartridge with a 100 × 800-µm detection window. The conditions used are detailed below. Inorganic salts (KNO3, KCl, KF, K2SO4, H3BO3) of analytical grade were purchased from Fluka. Na2CrO4?4H2O
JChemEd.chem.wisc.edu • Vol. 76 No. 6 June 1999 • Journal of Chemical Education
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In the Laboratory
was from Aldrich and the surfactant OFM-OH{ was from Waters. All solutions were prepared using HPLC-grade water from Aldrich.
Preparation of Buffer and Standards The buffer consisted of 4.6 mM chromate and 2.5 mM OFM-OH{ at pH 8 (adjusted with boric acid). Buffer was filtered with a 0.45-µm nylon filter. Such a solution can be stored three weeks in the refrigerator if sealed. Standards used for calibration curves were prepared at concentrations of 2, 5, 10, 15, 25, 50, 75, and 100 ppm, by diluting two stock solutions at 2 and 10 g/L according to Table 1. All solutions were degassed before use.
Table 1. Composition of Standard Solutions Solution, Anions Cl {, SO42 {, NO3{, F {
Under these analytical conditions, anions elute in the following order: chlorides, sulfates, nitrates, fluorides.
Laboratory Practical The laboratory session was divided into the following parts. Identification of anions. To determine the order of anion detection, a solution containing the 3 anions is injected, electrophoresed, and recorded. Then a minute quantity of one of the 3 anions is added to the previous solution and this solution analyzed. The two electropherograms thus obtained are superimposed. This spiking procedure identifies the corresponding peak on the electropherogram. This operation is repeated until all anions have been identified. Standards injection. Injections of standards are performed in triplicate in order of increasing concentration. Subsequently, these different injections are used to obtain a calibration curve (concentration versus peak area). It is possible to deduce a response coefficient for each anion under these conditions. Analysis of an unknown water. A triplicate injections of an unknown water sample are recorded. From the average peak area obtained from the electropherogram and with the calibration curve, anion concentrations are deduced. Results and Discussion A typical electropherogram is reported in Figure 5. Peak integration is automatically carried out by the Gold software. Data are valid only if the height of a peak is greater than 3 times the local background noise. Integrations are monitored for each electropherogram, so that the error rate is reduced, especially at low concentrations. Calibration curves are reported in Figure 6. In the concentration range 2–100 ppm, calibration curves are linear for all anions studied. Correlation coefficients are above .99, generally .999. The linear calibration curves do not exactly intersect the origin. This is a direct consequence of a weak residual background noise, which is very difficult to eliminate under the defined conditions. On the bases of the calibration curve slopes, fluoride is the most sensitive anion, followed by chloride, sulfate, and nitrate. 814
Concn/ppm
20 µL in 20 mL
→
50 µL in 20 mL
→
5
100 µL in 20 mL
→
10
75 µL in 10 mL
→
15
2
Stock Solution 10 g/L Cl {, SO42{, NO3{, F {
Experimental Conditions The parameters for an analytical run were: applied electric field: 10 kV hydrodynamic injection at 5 psi: 5 s temperature: 23 ± 0.1 °C electropherograms recorded for 5 min 1-min rinse period with chromate buffer between runs
Dilution
Stock Solution 2 g/L
25 µL in 10 mL
→
25
50 µL in 10 mL
→
50
75 µL in 10 mL
→
75
100 µL in 10 mL
→
100
Table 2. Results of Water Analyses Anion
Measured/ ppm ± SD
Supplier's Analysis/ppm
a. Commercial drinking water (av 11 runs) 6.0 ± 1.0
6
Cl{
22.9 ± 1.4
23
NO3{
16.8 ± 1.7
16
SO42{
b. Water from Yvette River (av 8 runs) SO42{
77.7 ± 2.1
NA
{
43.4 ± 2.5
NA
NO3{
20.3 ± 2.0
NA
Cl
Figure 5. Electropherogram of a 25-ppm standard mixture.
Figure 6. Calibration curves of the anions.
Journal of Chemical Education • Vol. 76 No. 6 June 1999 • JChemEd.chem.wisc.edu
In the Laboratory
From the calibration curves obtained, several concentrations of anions in mineral waters, drinking waters, and river waters can be measured. Fluoride was never detected. The average measured concentrations are given in Table 2a for a commercial drinking water (Cristalline brand) and in Table 2b for a river water (Yvette River water). For the commercial drinking water, the average measured concentrations were compared with the values given by the suppliers. Concentrations agree well with expected concentrations for sulfate, chloride, and nitrate. Conclusion A successful experimental capillary electrophoresis procedure has been developed to determine the concentration of chloride, sulfate, nitrate, and fluoride in water. It is particularly suited for the 2–100 ppm concentration range, which corresponds to the typical concentration of these anions in
drinking water. The main advantages of CE over ion HPLC are rapidity, simplicity, and efficiency. No sample preparation is necessary. Such an experimental procedure is suitable for a 4-hour laboratory training session. It can be extended to other anions, such as bromide, nitrite, phosphate, carbonate, and chlorate by adjusting a few parameters, especially the applied electric field and the dimension of the capillary. Literature Cited 1. Morin, P.; C. François, C.; Dreux, M. Analusis 1994, 22, 178– 187. 2. Rhemrev-Boom, M. M. J. Chromatogr. A 1994, 680, 675–684. 3. Oehrle, S. A.; Blanchard, R. D.; Stumpf, C. L.; Wulfeck, D. L. J. Chromatogr. A 1994, 680, 645–652. 4. Kuhn, R.; Hoffstetter-Khun, S. Capillary Electrophoresis: Principles and Practice; Springer: Berlin–Heidelberg, 1993.
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