Altering the selectivity of inorganic anion separations using electrostatic capillary electrophoresis

Abstract

A new method for altering the selectivity of inorganic anion separations in capillary electrophoresis is described. Addition of the zwitterionic surfactant 3-(N,N-dimethyldodecylammonio)propane sulfonate (DDAPS) to the background electrolyte modifies the migration order via electrostatic ion chromatography type interactions. Variation of the DDAPS surfactant concentration from 4 to 120 mM monotonically alters the selectivity from electrophoretic mobility based to that of electrostatic ion chromatography, without increasing Joule heating. This technique was applied to the determination of nitrate, nitrite, bromide and iodide in artificial seawater. Detection limits for the anions in 1∶5 diluted seawater were 11, 5, 7 and 11 μM, respectively.

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1 Introduction

The determination of small inorganic anions is important in a variety of fields such as the food, clinical and biological sciences, and also in environmental and industrial applications.¹ Although traditionally performed using ion chromatography,¹ capillary electrophoresis (CE) offers more efficient and rapid separations of these analytes.² A wealth of papers have been published on methods to alter the selectivity in CE.^3–7 Even considering the vast amount of work done (ref. 5 quotes 293 papers), the existing methods remain limited, especially for small inorganic anions. Introduction of secondary equilibria is one of the most effective means of altering selectivity in CE. However, complexation equilibria, such as those used with metal cations,^8–10 are generally not possible with anionic species. More commonly, acid–base equilibria or ion exchange equilibria are used. It is well known that selectivity changes will occur when the pK_a of the analyte is near that of the buffer. Thus, acid dissociation equilibria offer an effective means of altering the mobility of weakly acid species such as phosphate,¹¹ borate,² carbonate^2,12 and nitrite.^13,14 However, this approach is of little use for varying the mobilities of inorganic species such as chloride, bromide, nitrate, iodide and fluoride that possess unattainable pK_a values.¹⁵

Alkylammonium surfactants such as tetradecyltrimethylammonium bromide are commonly added to the buffer to reverse the electroosmotic flow (EOF) for anion separations.^2,12 In addition to acting as flow modifiers, it was noted that the concentration of alkylammonium surfactant affects the migration times of some inorganic anions through ion pairing interactions.^2,16,17 Studies of inorganic anion migration in micellar electrokinetic capillary chromatography demonstrated that anion migration was retarded by ion association with free surfactant monomers and distribution into the cationic micelles.¹⁶ It has also been suggested that the selectivity changes observed with the use of cationic surfactants result from ion exchange partitioning effects.^4,18,19

The ability to introduce an ion exchange element to CE separations is particularly powerful since these two techniques offer complementary selectivity.²⁰ Ion exchange selectivity may be more directly introduced into CE by the addition of polycationic species such as poly(1,1-dimethyl-3,5-dimethylenepiperidinium), poly(1,1-dimethyl-3,5-dimethylenepyrrolidinium) and poly(diallyldimethylammonium chloride).^21,22 The degree to which ion exchange retards anion mobility is directly related to the concentration of polyelectrolyte. Also, varying the electrolyte concentration in the background buffer changes the degree to which the analyte interacts with the polymer and its resultant migration time.²² These parameters allow the researcher to adjust the extent to which the separation occurs via electrophoretic or ion exchange effects.

Recently, electrostatic ion chromatography (IC) was introduced for the chromatographic separation of inorganic ions.²³ Electrostatic IC uses an octyldecylsilane column coated with a zwitterionic surfactant containing both anionic and cationic functional groups. Ions in solution cluster around the zwitterion charges and create an electrical double layer. Analyte ions experience a simultaneous attraction to and repulsion from this double layer and are distributed between the stationary phase and mobile phase. Retention is dependent on the propensity of the analyte to form neutral ion pairs with the double layer and are based on the polarizability of the ion, rather than on classical ion exchange selectivities. As a result, the order of elution for electrostatic IC differs dramatically from that of classical ion exchange chromatography or capillary zone electrophoresis.^20,24

This study was aimed at combining the speed and efficiency of CE with the entirely different selectivity offered by electrostatic IC. A zwitterionic surfactant is added directly to the electrophoretic buffer to alter the selectivity of inorganic anion separations. The concentration of zwitterion present dictates the resulting migration order of the anions since it determines the extent of electrophoretic or chromatographic influence on the separation. A particular advantage of using zwitterionic agents is that high concentrations can be used without affecting Joule heating.

This method was applied to the determination of iodide, nitrate, nitrite and bromide in artificial seawater. This separation is conventionally done by IC¹ and is one of the more difficult matrices to deal with in CE. A major challenge in quantifying species in seawater is the interference of matrix components such as chloride which hinder the reliability of analysis. In this work, we used our ability to alter the migration times and order of the anions to remove them from the area affected by matrix peaks.

2 Experimental

2.1 Apparatus

Experiments were performed using a P/ACE 2100 system (Beckman Instruments, Fullerton, CA, USA) with UV absorbance detection. Data acquisition (5 Hz) and control were performed using P/ACE Station software for Windows 95 (Beckman) on a Pentium 120 MHz microcomputer. Untreated silica capillaries (Polymicro Technologies, Phoenix, AZ, USA) with an inner diameter of 50 μm, outer diameter of 365 μm and total length of 47 cm (40 cm to the detector) were used.

2.2 Reagents

All solutions were prepared in Nanopure (18 MΩ) water (Barnstead, Chicago, IL, USA). Buffers were prepared from analytical-reagent grade orthophosphoric acid (BDH, Toronto, ON, Canada) with sodium hydroxide (BDH) to adjust the pH, or potassium chromate (BDH) with phosphoric acid (BDH) to adjust the pH. The pH values were measured using a Model 445 digital pH meter (Corning, Acton, MA, USA) calibrated immediately prior to use. The surfactants tetradecyltrimethylammonium bromide (TTAB) (Sigma, St. Louis, MO, USA) and 3-(N,N-dimethyldodecylammonio)propane sulfonate (DDAPS) (Aldrich, Milwaukee, WI, USA) were used as received. A 2 mM solution of mesityl oxide (Aldrich) in water was used as the neutral EOF marker. Previous studies have demonstrated that mesityl oxide is an appropriate EOF marker in micellar media.^25,26

Samples of 0.5 mM anion solutions in water were prepared from sodium nitrite (BDH), potassium nitrate (BDH), potassium bromide (Fisher Scientific, Fair Lawn, NJ, USA), sodium fluoride (Fisher Scientific), sodium chloride (BDH), sodium sulfate (Fisher Scientific), potassium oxalate (Matheson, Norwood, OH, USA), sodium iodide (BDH) and potassium thiocyanate (BDH) without further purification.

Artificial seawater was prepared by the method of Lyman and Fleming²⁷ with the exception that bromide ion was omitted and a 1∶5 dilution was made prior to analysis. The final concentrations were as follows: 80.4 mM NaCl (BDH), 4.90 mM MgCl₂ (BDH), 5.52 mM Na₂SO₄ (BDH), 1.50 mM CaCl₂ (Anachemia, Rouses Point, NY, USA), 1.78 mM KCl (BDH), 458 μM NaHCO₃ (BDH), 82.3 μM H₃BO₃ (BDH), 18.2 μM SrCl₂ (Fisher) and 15.0 μM NaF (Fisher). Standard dilutions of a stock standard solution of 5 mM NaI, KNO₃, KBr and NaNO₂ in artificial seawater were used to generate samples of the desired concentrations.

2.3 Procedures

New capillaries were pre-treated with a high pressure (138 kPa) rinse using 0.1 M NaOH for 5 min. Prior to each run, the capillary was rinsed at high pressure with 0.1 M NaOH for 1 min, H₂O for 1 min and buffer for 2 min. In all experiments, the capillary was thermostated at 25 °C.

2.4 Anion separations

For studies of the effect of concentration of cationic surfactant on selectivity, 5–150 mM TTAB was added to 10 mM phosphate buffer at pH 7.2. A mixture of 0.5 mM NaNO₂, KNO₃, NaI and KSCN was injected using low pressure (3.45 kPa) for 1.0 s. The applied potential was −20 kV and the detector rise time was 1.0 s. The direction of the EOF was reversed when using a cationic surfactant (i.e., from the cathode to the anode) so that the anions were separated in the co-EOF mode and detected using direct UV detection at 214 nm. Standard additions of each analyte were performed at each concentration to determine the migration order. Duplicates of each run were performed.

For studies of the effect of concentration of zwitterionic surfactant on the selectivity, 3.75–120 mM DDAPS was added to 5 mM chromate buffer at pH 8.0. A mixture of 0.5 mM NaNO₂, KNO₃, KBr, NaI, KSCN, NaF, NaCl, Na₂SO₄ and K₂C₂O₄ in water was separated. The direction of the EOF was from the anode to the cathode so that anions were separated in the counter-EOF mode and detected at the anode using indirect UV detection at 254 nm. All other conditions were the same as above.

For the separation of anions in seawater, 30 mM DDAPS was added to 10 mM phosphate buffer (pH 8.0) to separate KBr, KNO₃, and NaNO₂ with a 3 s low pressure sample injection. DDAPS (10 mM) was used to separate NaI, KNO₃ and NaNO₂ using a 1 s sample injection. Both separations were done in counter-EOF mode using an applied voltage of −20 kV and direct detection at 214 nm. All other conditions were the same as for previous separations.

2.5 Mobility measurements

The apparent mobility, μ_app, of the anions was determined from the migration time under constant voltage conditions using the equation: where L_T is the total capillary length, L_d is the effective capillary length to the detector, t is the migration time and V_appl is the applied voltage.

The EOF was measured in the TTAB solutions by the conventional method of injecting a neutral marker, namely mesityl oxide, for 1 s using low pressure. A constant voltage of 20 kV was applied for 7.5 min and the neutral compound was eluted from the capillary with the generated EOF. Direct detection was used at 254 nm. Eqn. (1) was applied to calculate the EOF by inserting the migration time of the neutral marker.

The EOF generated in the presence of zwitterionic surfactants is slow. As a consequence, conventional measurement is impractical owing to the length of time required to elute a neutral marker. In this work, the EOF was measured in DDAPS solutions using the three-peak injection method of Williams and Vigh.²⁸ In this procedure, the capillary was first rinsed and filled with the buffer. The EOF marker, water for indirect detection, was injected using low pressure for 1.0 s. This band was pushed through the capillary using low pressure for 0.5 min. A second water sample was introduced as before and another low pressure push was applied for 0.5 min. A constant voltage of 10 kV was then applied for 99 s. The position of the two neutral marker peaks was altered by the resultant EOF. A final water peak was injected and the bands were swept from the capillary by applying low pressure for 15 min. Indirect detection was performed at 254 nm. The EOF was finally calculated using the equation²⁸

where t₁, t₂ and t₃ are the times required to push bands 1, 2 and 3, respectively, past the detector, L_T is the total length of the capillary, L_d is the effective capillary length to the detector, t_inj is the injection time, V_appl is the applied voltage and t_migr is the time of applied voltage. A voltage ramp correction was not done as its effect was insignificant.

Finally, the effective mobility of the analytes, μ_eff, was calculated using the equation

μ_eff = μ_app − μ_EOF (3)

2.6 Quantification of anions in seawater

Limits of detection of the anions in seawater were determined using a procedure based on the US Environmental Protection Agency methodology.²⁹ This approach determines the minimum amount of sample that can be reported to be greater than the background noise (blank run) with 95% confidence. Using the experimental conditions described in Section 2.4, a calibration curve was generated over the range 0.05–1.0 mM using dilutions of the 5 mM stock standard solution of the anions. Then, a 75 μM sample of NaNO₂, KNO₃ and KBr (about 5–10 times the estimated detection limit) in 5∶1 diluted seawater was separated nine times using 30 mM DDAPS in 10 mM phosphate (pH 8.0). The standard deviation for these replicate injections was determined. The detection limit is the standard deviation multiplied by the Student t value. For n − 1 or 8 degrees of freedom, the t value for the one-sided 95% confidence interval is 1.860. The same procedure was used to determine iodide detection limits using 10 mM DDAPS in 10 mM phosphate (pH 8.0).

3 Results and discussion

3.1 Effect of TTAB concentration on anion selectivity

TTAB is a cationic surfactant commonly used to reverse the EOF in separations of inorganic and small organic anions.^2,12 Selectivity changes with increasing TTAB concentration have been reported. Jones and Jandik² described a change in migration order for bromide, chloride, nitrite and sulfate with the use of up to 5 mM TTAB in chromate. Jimidar and Massart¹⁹ also showed that bromide crosses over chloride when using up to 1 mM TTAB. These anion selectivity changes with TTAB concentration result from the electrostatic ion pairing between the negatively charged analyte and positively charged surfactant monomers and/or micelles.^16,17,30

We wished to investigate whether more dramatic selectivity changes could be achieved by using higher concentrations of TTAB. However, a number of difficulties were experienced. First, TTAB is insoluble in chromate, the most common probe for inorganic anions, at pH <8.0,¹¹ which makes TTAB unsuitable for indirect detection at low pH. Second, severe baseline disturbances (data not shown) were observed when high concentrations of TTAB (>5 mM) were added to the chromate buffer. Third, as shown in Fig. 1, increasing the concentration of TTAB significantly increased the current, and thus the Joule heating.

Fig. 1 Effect of type and concentration of surfactant on current. Experimental conditions: applied voltage, −20 kV; temperature, 25 °C; capillary, 47 cm (40 cm to detector) × 50 μm id; buffer, 5.0–150 mM TTAB in 10 mM phosphate (pH 7.2) and 5.0–100 mM DDAPS in 10 mM phosphate (pH 7.2)

Given these restrictions we were only able to investigate the effect of the concentration of cationic surfactant on the migration of anions that were UV active. Fig. 2 shows the effective mobilities (Section 2.5) of these anions at various concentrations of TTAB. Dramatic changes in mobility were observed up to ∼15 mM TTAB. However, no significant mobility changes were observed at higher concentrations. This transition is due to the complex nature of the surfactant additive. As the concentration of TTA⁺ increases, the number of ion association sites increases. At the same time, the concentration of bromide, a potential eluent, increases proportionally whereas the concentration of the phosphate buffer, a stronger eluent, stays constant at 10 mM.³¹ Thus, as the TTAB concentration increases in Fig. 2, the anions become increasingly retained by the cationic surfactant as its concentration increases since the phosphate concentration relative to cationic surfactant decreases. At higher concentrations of TTAB, bromide becomes the more concentrated eluent, but the ratio of eluent to retentive sites remains constant. This provides a constant amount of eluent to compete with the analytes for ion association sites and thus the effective mobilities of the analytes do not change. This illustrates that the use of TTAB provides some mobility alteration, but problems restrict its use to a narrow set of experimental conditions.

Fig. 2 Effect of concentration of cationic surfactant on the selectivity of inorganic anionic separations. Experimental conditions: buffer, 10 mM phosphate and various concentrations of TTAB (pH 7.2); sample, 0.5 mM of each anion; 1 s hydrodynamic injection. Other conditions as in Fig. 1

3.2 Effect of DDAPS concentration on anion selectivity

The zwitterionic surfactant, DDAPS was added to a chromate buffer to investigate its effect on the selectivity. In contrast to TTAB, Fig. 1 shows that the current remains constant over the full DDAPS concentration range, allowing high concentrations of surfactant to be used if desired. The use of a zwitterionic species does not contribute to the conductance of the buffer since its net charge is zero. As a result, the current remains constant as the concentration increases, inducing no additional Joule heating. Fig. 3 shows the effective mobilities (Section 2.5) of NO₂⁻, NO₃⁻, Br⁻, I⁻, SCN⁻, F⁻, Cl⁻, SO₄²⁻ and C₂O₄²⁻ determined in 3.75–120 mM DDAPS. When the zwitterionic surfactant is used, a dramatic change in selectivity is observed for several analytes. In particular, more polarizable anions such as thiocyanate, iodide, nitrate and bromide exhibit substantial retarded mobility as the concentration of surfactant increases. This is consistent with the retention observed in electrostatic IC.^24,32,33 Furthermore, no leveling off behavior is observed in Fig. 3, in contrast to the TTAB behavior shown in Fig. 2. This is due to using zwitterionic surfactant where it is possible to add the retentive phase (surfactant) without increasing the eluent strength. In contrast, the cationic surfactant must always be accompanied by a counter-ion which will act as an eluent for the ion exchange retention.

Fig. 3 Effect of concentration of zwitterionic surfactant on the selectivity of inorganic anion separations. Experimental conditions: buffer, 5.0 mM chromate and various concentrations of DDAPS (pH 8.0). Other conditions as in Fig. 1

At low concentrations of DDAPS, the mobility order is Br⁻, Cl⁻, I⁻, SO₄²⁻, NO₂⁻, NO₃⁻, C₂O₄²⁻, SCN⁻ and F⁻, as shown in Fig. 4(A). This is the same as that observed for low concentrations of TTAB.³⁴ However, at 120 mM DDAPS, the mobility order is SO₄²⁻, Cl⁻, C₂O₄²⁻, NO₂⁻, Br⁻, F⁻, NO₃⁻, I⁻ and SCN⁻. This differs significantly from conventional capillary zone electrophoresis. Moreover, this separation order is essentially identical with that observed in electrostatic IC (SO₄²⁻ ≈ Cl⁻, NO₂⁻, Br⁻, NO₃⁻, ClO₃⁻, I⁻ and SCN⁻).²⁴ A typical electropherogram for the separation of inorganic anions using 60 mM DDAPS is shown in Fig. 4(B).

Fig. 4 Typical electropherograms for the separation of inorganic anions using DDAPS. Peaks: (1) bromide; (2) chloride; (3) iodide; (4) sulfate; (5) nitrite; (6) nitrate; (7) oxalate; (8) thiocyanate; (9) fluoride. Experimental conditions: buffer, 5.0 mM chromate and 3.75 and 60 mM DDAPS (pH 8.0). Other conditions as in Fig. 1

3.3 Mechanism of retention

The experimental conditions used in this study are similar to those used in electrostatic IC. The zwitterionic surfactant DDAPS used here is the same surfactant as used to coat an ODS column in electrostatic IC. Also, both techniques provide retention of inorganic anions, with the polarizable anions being more strongly retained. Furthermore, the migration order observed in this study is identical with the elution order observed in electrostatic IC.²⁴ These factors lead us to believe that the mechanism for electrostatic IC also explains the behavior observed here. In electrostatic IC, cations and anions in solution are retained by the positively and negatively charged groups of the zwitterionic surfactant to form an electrical double layer. Simultaneous electrostatic attraction and repulsion of the analyte anions with the zwitterionic electrical double layer causes anion retention and separation. The propensity of the analyte ion to form ion pairs with the double layer dictates how long it will be retained.²⁴ The more polarizable anions are retained longer since they can form stronger ion pairs with the electrical double layer. This explains why thiocyanate, iodide, bromide and nitrate are more strongly retained as the concentration of DDAPS increases.

It is further thought that anions interact with the micelles primarily in bulk solution since the peak shapes are generally symmetric. Interactions with the micelles at the walls would cause tailed peaks due to resistance to mass transfer of analyte from the bulk solution to and from the capillary wall. These tailed peaks are evident in open tubular capillary electrochromatography and still remain one of its disadvantages.³⁵

3.4 Determination of anions in seawater

Capillary zone electrophoresis works best with low conductivity samples. As a result, seawater has posed a challenging matrix for CE. First, the high conductivity of seawater causes electrodispersion within the sample zone, resulting in severe distortion and broadening of the analyte peaks. Second, the mobility of chloride is similar to that of anions of interest, making separation difficult. Previous CE separations of bromide, iodide, nitrate, nitrite and thiocyanate were performed using chloride as the electrolyte to reduce matrix effects in direct UV detection.³⁶ Recently, Fukushi et al.³⁷ used artificial seawater as the electrolyte solution in CE to determine bromide, nitrite and nitrate in seawater. However, the high current generated, 330 μA, is a drawback. Electrostatic IC has also been demonstrated to be effective for the determination of bromide, nitrate and iodide in seawater by using dilute seawater as the eluent.³⁸ Therefore, we will explore the use of electrostatic capillary electrophoresis for the determination of anions in seawater.

The seawater matrix was diluted 1∶5 to minimize electrodispersion in the sample zone caused by high amounts of chloride. Based on Fig. 3, 10 mM DDAPS should separate nitrate, nitrite and iodide from the seawater matrix, while still eluting iodide in a reasonable time. Fig. 5(A) shows the separation of NO₂⁻, NO₃⁻ and I⁻ in 1∶5 diluted seawater. Calibration graphs for all anions were linear from 0.05 to 1.0 mM. Table 1 lists the detection limits, correlation coefficients (r²) and RSD values for the migration times and peak areas. Timerbaev et al. achieved detection limits of 1–2 μM in 1∶10 diluted seawater at 200 nm using a chloride background electrolyte.³⁶

Fig. 5 Determination of inorganic anions in artificial seawater. Experimental conditions: (A) buffer, 10 mM DDAPS in 10 mM phosphate (pH 8.0); sample, 150 μM of each anion in diluted artificial seawater; 1 s hydrodynamic injection; (B) buffer, 30 mM DDAPS in 10 mM phosphate (pH 8.0); sample, 75 μM of each anion in diluted artificial seawater; 3 s hydrodynamic injection. Other conditions as in Fig. 1

Table 1 Determination of inorganic anions in artificial seawater

Anion	Correlation coefficient (r^2)	Peak area RSD (%)	Migration time (min) RSD (%)	Detection limit in 1∶5 diluted seawater/μM
a Experimental conditions as in Fig. 5(A) except that a 100 μM sample was used. b Experimental conditions as in Fig.5(B).
Iodide^a	0.955	10.3	0.64	11
Nitrate^b	0.991	7.2	0.74	11
Nitrite^b	0.992	3.1	0.95	5
Bromide^b	0.990	3.5	0.82	7

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Bromide could not be determined using the conditions in Fig. 5(A) as it still co-eluted with the large chloride matrix peak and could not be detected. If, instead, 30 mM DDAPS was added to the phosphate buffer, the bromide could be removed from the chloride matrix, as shown in Fig. 5(B). The ability to move peaks selectively demonstrates the flexibility of this technique. However, this higher concentration of DDAPS results in excessively long migration times for iodide.

The addition of zwitterionic surfactants to the background electrolyte allows one to change the selectivity of inorganic anion separations from that of capillary zone electrophoresis to the completely different selectivity of electrostatic IC. The choice of zwitterionic surfactant concentration allows the researcher to decide the migration order of the analytes, minimizing the impact of interfering peaks, with the added bonus of not inducing any additional Joule heating.

Acknowledgements

This work was supported by the Natural Sciences and Engineering Research Council of Canada and by the University of Alberta.

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