Sensitive arsenic speciation by capillary electrophoresis using UV absorbance detection with on-line sample preconcentration techniques

Talanta 181 (2018) 366–372

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Sensitive arsenic speciation by capillary electrophoresis using UV absorbance detection with on-line sample preconcentration techniques Ho Gyun Lee, Joon Yub Kwon, Doo Soo Chung

T

⁎

Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea

A R T I C L E I N F O

A B S T R A C T

Keywords: Capillary electrophoresis Arsenic speciation Counter flow electrokinetic supercharging Isotachophoresis Sample stacking technique

The World Health Organization (WHO) guideline states that the total arsenic concentration in drinking water must not exceed 10 ppb. However, arsenic toxicity varies significantly, with inorganic arsenic species being more toxic than organic species. Arsenic speciation is therefore important for evaluating the health risks from arseniccontaminated drinking water. Capillary electrophoresis provides the necessary high performance separation to determine arsenic species in water, but its sensitivity with absorbance detection is far below than needed. Using a coated capillary, several on-line sample preconcentration techniques such as large volume sample stacking with an electroosmotic flow pump, field amplified sample injection (FASI), transient isotachophoresis (tITP), electrokinetic supercharging (EKS) combining FASI and tITP, and counter flow (CF)-EKS, were therefore investigated. With CF-EKS using phosphate and N-cyclohexyl-2-aminoethanesulfonate as leading and terminating electrolytes, respectively, standard samples of arsenite, arsenate, monomethylarsonic acid, and dimethylarsinic acid were preconcentrated from 6,300- to 45,000-fold. The limits of detection obtained with UV absorbance detection were 0.08–0.3 ppb As. For a spring water sample spiked with the four arsenic species, LODs of 2–9 ppb As were obtained, which are lower than the WHO guideline of 10 ppb total As.

1. Introduction More than 50 arsenic (As) compounds of varying toxicities exist naturally [1]. Inorganic As compounds generally being more toxic than organic As compounds, some of which are nontoxic [2,3]. However, the majority of current As regulations focus on the total As content [4–6]. For example, the World Health Organization (WHO) guideline for drinking water is < 10 ppb total As [7]. It is partly due to practical limits of detection (LODs) obtained with usual As speciation techniques, although HPLC-inductively coupled plasma/mass spectrometry (ICP/ MS) of high cost, for example, can deliver LODs of As species lower than 40–60 ppt [8]. Therefore there is a need to develop sensitive, but readily accessible, As speciation methods [9–12]. Capillary electrophoresis (CE) is a suitable high performance separation technique for As speciation. However, the small separation capillary dimensions result in the LODs for As species being significantly higher than the WHO guideline when UV absorbance detection is employed [13]. Potential solutions for this issue include incorporating more sensitive detection methods [14–16] and sample preconcentration techniques [4,17,18]. In addition, the combination of two or more techniques has also been studied to further enhance the sensitivity [4,19,20].

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To improve the As speciation sensitivity of CE, we compared on-line sample preconcentration techniques for CE after sample injection into the capillary. Large volume sample stacking with an electroosmotic flow (EOF) pump (LVSEP) [21], field amplified sample injection (FASI) [22], and transient isotachophoresis (tITP) [23] allowed As analyte concentrations to multiply by tens to hundreds of times. To further improve the sensitivity, electrokinetic supercharging (EKS) [24], which combines FASI and tITP, was applied. A hydrodynamic counter flow (CF) was then applied during the FASI process to counterbalance the movement of the injected sample plug in order to increase the injection quantity and to secure an adequate capillary length for tITP [25]. For standard samples, CF-EKS yielded 6300–45,000-fold sample enrichments with LODs (S/N = 3) of 0.08–0.3 ppb As using the built-in UV detector of a commercial CE instrument. For a sample of spring water containing As exceeding the WHO guideline, CF-EKS yielded enrichments of 230–1200-fold with LODs of 2–9 ppb As, which are sufficient to confirm, for the first time in several decades, that CE-UV with an online sample preconcentration is suitable for As-containing water analysis.

Corresponding author. E-mail address: [email protected] (D.S. Chung).

https://doi.org/10.1016/j.talanta.2018.01.034 Received 16 October 2017; Received in revised form 10 January 2018; Accepted 12 January 2018 0039-9140/ © 2018 Elsevier B.V. All rights reserved.

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2.2. Instruments and methods A PA 800 Plus instrument from Sciex (Framingham, MA, USA) was utilized for all electrophoretic processes. A μSiL-FC coated fused silica capillary of 60 cm (50 μm id, 365 μm od, and 50 cm distance to the detector) was purchased from Agilent (Santa Clara, CA, USA). Between runs, the capillary was rinsed with deionized water at 30 psi for 15 min and then with the run buffer at 30 psi for 10 min. A voltage of −20 kV was applied for separation and the As analytes were monitored at 200 nm. The temperature was set at 25 °C. Instead of the peak heights from capillary zone electrophoresis (CZE), the plateau heights of a 75ppm As sample in 100 mM sodium phosphate injected at 15 psi for 10 s and then pushed at 2 psi were used as references to calculate the enrichment factors (EFs) of on-line sample preconcentration techniques. It was to avoid the dependence of the peak heights on the injection volume. This was rather significant especially when a small amount of the sample was injected. The total As content in a sample of water from a local spring known to be contaminated with As was determined by ICP/MS (Varian 820MS, Analytik Jena, Jena, Germany) in order to compare with our method. To take care of the matrix effects of the spring water sample, standard addition methods were applied.

Fig. 1. Structures of arsenic compounds and their pKa values [49,50].

2. Materials and methods 2.1. Materials and sample preparation Sodium phosphate dibasic (99%), N-cyclohexyl-2-aminoethanesulfonate (CHES, 99.5% and 99.99%), NaOH (99.99%), sodium arsenate dibasic heptahydrate [As(V)], sodium (meta)arsenite [As(III)], dimethylarsinic acid (DMA), and disodium methyl arsonate hexahydrate (MMA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). NaOH (97%) was obtained from Daejung Chemical (Siheung, Korea). The fluorocarbon surfactant FC-4430 was from 3 M (St. Paul, MN, USA), and deionized water was prepared using a Lab Tower EDI 15 UV system from Thermo Fisher Scientific (Waltham, MA, USA). The CE run buffers consisted of various concentrations of sodium hydrogen phosphate titrated to pH 9.6 using 1 M NaOH, which were also employed as leading electrolyte (LE) solutions. In addition, CHES buffers of various concentrations titrated to pH 9.6 using 1 M NaOH were employed as terminating electrolyte (TE) solutions. All the buffer solutions were set at pH 9.6 in order to ionize the four As species in Fig. 1 including As(III) with pKa 9.23 and not to harm the inner wall coating of a coated capillary used in our investigation (see below). All solutions (with the exception of the sample solutions) also contained 0.02 wt% FC-4430 to enhance the durability of the inner wall coating. Stock solutions (100 mM) containing each As species were prepared in deionized water, transferred to Parafilm-sealed vials to prevent CO2 contamination from the air, and stored in the dark at 4 °C. Standard samples for LVSEP and FASI were prepared in diluted run buffer solutions. For tITP, EKS, and CF-EKS, standard samples were prepared in CHES buffer solutions while a CHES buffer was added to the spring water sample for CF-EKS.

3. Results and discussion 3.1. Coated capillary for arsenic speciation Various schemes of As speciation with CE have explored for several decades, as summarized in Table 1. Efforts were made to improve the sensitivity of CE with the most widely used UV detection due to the low absorbance of As species. Among many As species, the four highly toxic ones predominantly investigated as in this report are acidic and their anions at high pH are highly mobile. In order to analyze the highly mobile anions under an electric field of normal polarity, it is desirable to have a high electroosmotic flow. Hence, basic run buffers of low ionic strength were commonly employed for a bare fused silica capillary [18,26,27]. Then it is rather difficult to apply sample preconcentration techniques based on the difference in conductivities between the sample and run buffer zones, such as most widely used simple sample stacking and large volume sample stacking (LVSS). Thus either more

Table 1 Comparison of As speciation with CE. Preconcentration-Detection

Capillary

Background electrolyte

LOD

Ref

UV

Bare

19.2–30 ppm

[13]

UV

PDDAC dynamic coating

100–460 ppm

[18]

SDMEa-UV

Bare

8–53 ppb

[17]

FASI-UV

SMIL dynamic coating

20–70 ppb

[28]

Dynamic pH junction-UV LVSS-UV

Bare

25 mM phosphate pH 8 20 mM phosphate pH 10 15 mM phosphate pH 10.6 10 mM 2,6pyridinedicarboxylic acid pH 10.3 15 mM phosphate pH 10.6 20 mM carbonate pH 10 1.5 mM fluorescein pH 9.8 5 mM formic acid pH 2.95 20 mM phosphate pH 10 100 mM phosphate pH 9.6

195–292 ppb

[46]

31–100 ppb

[19]

60–160 ppb

[15]

0.1–1 ppm (organic) 65–250 ppm (inorganic) 20–100 ppt (inorganic)

[47]

Bare

b

Bare

c

Bare

ICP-MS

Bare

CF-EKS

μSiL-FC coated

LIF (indirect) MS

a b c

Single drop microextraction. Laser induced fluorescence. Mass spectrometry.

367

0.08–0.3 ppb

[48] Present method

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3.2. Large volume sample stacking using an EOF pump (LVSEP)

100

80 LVSEP current

The most widely used on-line sample preconcentration method for CE is field amplified sample stacking [29]. In this method, analytes dissolved in a low conductivity sample matrix are stacked at the boundary with a high conductivity run buffer. To obtain a high EF, the conductivity ratio and/or the amount of injected sample should be increased. However, due to the EOF mismatch and the reduced separation capillary length, the actual EF limit is about ten [30]. To overcome this, LVSS [31] that removes the sample matrix plug after sample stacking can be employed to increase the EF into the hundreds. LVSEP, a very convenient and efficient LVSS technique that does not require polarity switching, can be applied when the magnitude of the EOM is smaller than that of the analyte's electrophoretic mobility and its sign is the opposite of the analyte's mobility sign. For anionic analytes, a large volume of a low conductivity sample is injected and a voltage of reversed polarity is applied. In a capillary with a reversed EOF, cations can be enriched by LVSEP in a similar manner [32]. For LVSEP, a reduction in the EOM magnitude below that of the analyte mobilities is thus required. Among the various methods available [30,33–35], we selected to use a μSiL-FC coated fused silica capillary. Fig. 2b shows an LVSEP electropherogram of a 3.75 ppm standard sample dissolved in 0.1 mM sodium phosphate buffer, which was injected into the entire capillary, and then stacked and separated at −20 kV. The sharp absorbance increase at ~1.4 min indicated the passage of the 100 mM sodium phosphate run buffer zone from the outlet vial through the detection window. As the capillary got filled with the high conductivity run buffer from the outlet vial, the low conductivity sample matrix was expelled from the capillary, resulting in an increase in electric current (dotted line) and a decrease in the overall EOF. When the EOF and electrophoretic velocities were balanced, the stacked analytes switched the migration direction toward the detector. After complete removal of the matrix plug, the current reached 90 μA, which was close to the value for a capillary filled with the run buffer alone, and the electric field was distributed evenly over the capillary. This change accounted for the slight increase in absorbance at ~7.6 min, which correlated well with the migration time differences, e.g., between 14.0 min and 6.4 min for As(V) from LVSEP and CE, respectively. A standard sample prepared in the 1000-fold diluted run buffer was fully injected to achieve 130–180-fold enrichments with

60

2 60

4 40

1

20

Current (μA)

Absrobance (mAU)

80

40

(b) LVSEP, 3.75 ppm

20

3 1

2

4 (a) CZE, 75 ppm

0

0 0

5 10 15 Migration Time (min)

20

Fig. 2. (a) CZE of 75 ppm As of each As species [1; As(V), 2; MMA, 3; As(III), 4; DMA] in deionized water injected at 0.5 psi for 5 s, and (b) LVSEP of 3.75 ppm of each As species in 0.1 mM sodium phosphate buffer with full injection; 50/60 cm μSiL-FC coated fused silica capillary (50 μm id, 365 μm od); run buffer of 100 mM sodium phosphate (pH 9.6); −20 kV at 25 °C. Absorbance detection at 200 nm. Dotted line: electric current.

sensitive detection schemes such as CE-MS and CE-ICP/MS, or dynamic pH junction of a modest sample enrichment power were used to improve the detection sensitivity for the As species. On the contrary, with a coated capillary of a reduced or reversed electroosmotic mobility (EOM), a run buffer of high ionic strength could be used for better separation and higher sample enrichments based on the conductivity differences such as FASI [28] and LVSS with polarity switching [19]. Using a coated capillary of reduced EOM, we carried out a more exhaustive investigation on the sample preconcentration techniques for sensitive As speciation with CE. The EOM for a μSiL-FC coated fused silica capillary with a 100 mM sodium phosphate run buffer at pH 9.6 was estimated to be 1.68 × 10−8 m2/V s from the migration time of a neutral marker of 0.1 vol% DMSO. This is approximately a one third reduction from 4.87 × 10−8 m2/V s for a bare fused silica capillary. Fig. 2a shows that the four As species were separated well using the 100 mM phosphate run buffer with the coated capillary at a reverse voltage of −20 kV.

Table 2 Comparison of on-line sample preconcentration techniques for standard samples. ma

Mode

CZE

tITP

FASI

EKS

CF-EKS

a

As(V) MMA As(III) DMA As(V) MMA As(III) DMA As(V) MMA As(III) DMA As(V) MMA As(III) DMA As(V) MMA As(III) DMA

1.92 3.89 1.05 3.13 1.32 2.24 7.11 1.96 4.98 7.11 9.60 3.56 1.80 1.83 7.61 9.60 7.61 6.58 6.22 1.47

r2

−2

× × × ×

10 10−2 10−1 10−2

× × × × × × × ×

102 102 101 101 102 102 102 102

EF

– – – – 60 50 60 60 300 210 100 120 8200 4200 700 2600 45,000 18,000 6300 7100

0.998 0.997 0.992 0.999 0.995 0.999 0.998 0.991 0.984 0.996 0.972 0.993 0.995 0.994 0.991 0.999 0.993 0.998 0.994 0.998

%RSD Migration time

Height

0.1 0.2 0.1 0.2 0.1 0.2 0.2 0.2 0.2 0.1 0.3 0.1 0.2 0.1 0.3 0.1 0.2 0.2 0.2 0.3

9.0 9.4 7.5 8.0 7.4 8.2 6.6 7.1 6.0 5.8 7.0 10 7.2 7.0 7.7 9.7 7.6 8.3 9.3 9.7

Calibration curve slope. y = mx + b a) Slope between y: peak height (μAU) and x: sample concentration (ppb As).

368

LOD (ppb As)

3900 1900 710 2400 80 40 10 50 10 8 6 20 0.5 0.5 1.1 0.9 0.08 0.1 0.1 0.3

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arranged into zones in order of mobility. For analytes of trace amounts, the analytes are focused into peaks [38]. tITP combines ITP and CE in the same capillary. Among many tITP schemes [39], we chose the phosphate in the run buffer as an LE and CHES as a TE in the sample and/or in the plug inserted after the sample. The CHES buffer of pH 9.6 was also used to dissolve the As analytes as anions [40]. After an As sample prepared in the CHES (TE) buffer was injected into a capillary filled with the phosphate (LE) run buffer, the capillary was placed between two run buffer vials. Upon application of an electric field, ITP stacking proceeded initially as the zones in order of mobility (LE > As analytes > TE) were formed. Over time, the LE from the inlet vial passed the TE zone, thus breaking the ITP condition and resulting in CE of the concentrated analytes. One advantage of the ITP stacking scheme over LVSEP is that a low conductivity matrix is not required for the sample. As the ITP stacking effect depends on the LE concentration, we varied the phosphate run buffer concentration between 50 and 200 mM to compare the EF values obtained for a 7.5 ppm As sample prepared in a 50 mM CHES buffer and injected at 0.5 psi for 300 s. The EF values increased to 20–50 until the LE concentration reached 100 mM, after which little variations in the EF were observed although migration times were elongated. Consequently, we selected a 100 mM sodium phosphate buffer as both the LE and run buffer solution for tITP. When samples at lower concentrations were employed, however, a number of unknown peaks interfered with the analyte peaks. The peak heights of the unknown peaks were proportional to the CHES concentration, indicating that they originated from impurities in the CHES buffer. Although the impurity peaks were reduced by lowering the CHES concentration, the ITP performance was adversely affected at CHES concentrations < 2.5 mM. To resolve this issue, we inserted a concentrated TE plug following injection of the sample in a dilute CHES buffer. Compared to the sample prepared using 10 mM CHES buffer in the absence of a TE plug, the impurity peak heights decreased by ~10 times when the sample was prepared in 1 mM CHES buffer by inserting a 100 mM CHES buffer plug. No significant differences in analyte peak heights were observed when the TE plug length and concentration were within 2–10% of the total capillary length and 100–150 mM, respectively. Below these limits, the sample concentration effect was reduced due to insufficient ITP stacking [41]. The optimal condition was determined to be a 100 mM CHES buffer plug injected at 2 psi for 7.5 s following sample injection. Thereafter, the sample injection volume was optimized. Upon injecting a large amount of sample to enhance the ITP stacking effect, the remaining capillary length for the subsequent CE separation became insufficient. With sample injections < 21% of the total length, the EFs increased to 51–63. However, upon increasing the sample injection to > 24% of the capillary length, the DMA peak overlapped with the large CHES peak. The optimal conditions for the tITP of a standard sample were a 100 mM sodium phosphate buffer used as a run buffer, a sample dissolved in 1 mM CHES buffer injected into 21% of the total capillary length (15 psi for 10 s), a TE plug of 100 mM CHES buffer injected at 2 psi for 7.5 s, and separation at −20 kV. Fig. 3b shows that these conditions yielded EFs and LODs of 50–60 and 10–80 ppb As, respectively (see Table 2).

(a) FASI, 750 ppb

Absrobance (mAU)

3 1

60

4

2

3 40

(b) tITP, 7.5 ppm 2 1

20

4

0 0

5 10 15 Migration Time (min)

20

Fig. 3. (a) FASI of a 750 ppb As sample in 1 mM sodium phosphate buffer at −10 kV for 24 s, and electrophoresis at −20 kV. (b) tITP of a 7.5 ppm As sample in 1 mM CHES buffer injected into 21% of the total capillary length (15 psi for 10 s), a TE plug of 100 mM CHES buffer injected at 2 psi for 7.5 s, and electrophoresis at −20 kV. Others as in Fig. 2.

LODs of 7–26 ppb As (see Table 2), which are close to the WHO drinking water guideline. Compared with the EF of 34–62 and the LODs of 31–100 ppb As obtained from LVSS with polarity switching using a bare fused silica capillary [19], our LVSEP results obtained with a coated capillary were more than four times better in addition to the convenience of the automatic LVSEP operation. However, considering that environmental samples will usually exhibit a higher conductivity, thus lowering the EFs obtainable with LVSEP, further improvements in sample enrichment are necessary. 3.3. Field amplified sample injection (FASI) FASI, also called as field enhanced sample injection [28], is a sample preconcentration technique employed during the electrokinetic injection (EKI) of a low conductivity sample into a capillary filled with a high conductivity run buffer. A large sample amount can be injected and stacked at the conductivity boundary. For FASI of anions, the sample conductivity should be lower than that of the run buffer, and either a coated capillary or the injection of a water plug must be employed prior to sample injection to overcome the EOF [36,37]. The use of a coated capillary allows for simple operation, yet the pH range is governed by the coating stability. The μSiL-FC coated fused silica capillary employed in this report has an operational range of pH 2.5–10. As the FASI voltages were varied from −2 to −15 kV, the FASI times were adjusted to inject equal sample amounts into the capillary filled with 100 mM sodium phosphate run buffer. No significant differences in peak areas were observed up to −10 kV, although some degree of peak broadening was observed at −15 kV. Then as the FASI time was increased at −10 kV, the peak heights were increased but peak broadening was observed at FASI times > 24 s. Fig. 3a shows an electropherogram of a 750 ppb As in 1 mM sodium phosphate buffer with the optimal FASI at −10 kV for 24 s, yielding 100–300-fold enrichments. LODs were 6–20 ppb As (see Table 2). These results were better than previously reported 20–70 ppb obtained from FASI with indirect UV detection using a successive multiple ionic polymer layer coated capillary [28]. However, for environmental samples of higher conductivity, further improvements in sample enrichment are necessary.

3.5. Electrokinetic supercharging (EKS) EKS combines two sample preconcentration techniques; FASI during injection and tITP after injection [24]. When EKS was first developed, an LE plug was injected into a capillary filled with a run buffer, a low conductivity sample was introduced by FASI, and then a short TE plug was injected for the subsequent tITP [24]. The EKS procedure was later simplified by adding a TE to the sample instead of inserting a TE plug after the FASI step [42]. However, we employed the TE plug method due to the impurity issues described in the previous section. In the FASI of Section 3.3, the injection time was limited to 24 s at

3.4. Transient isotachophoresis (tITP) In isotachophoresis (ITP), a sample plug is placed between fast LE and slow TE zones. When an electric field is applied, the analytes are 369

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(a)

Fig. 4. (a) EKS of 750 ppb As in 1 mM CHES buffer. (b) EKS of 7.5 ppb As in 1 mM CHES buffer. Upper electropherogram obtained using 99.5% CHES and 97% NaOH and lower one with less impurity peak interference using 99.99% CHES and 99.5% NaOH. EKS conditions; FASI at −20 kV for 1 min, a TE plug of 100 mM CHES buffer injected at 1 psi for 1 min, 30 s waiting, and tITP at −20 kV. Others as in Fig. 2.

(b) 80

8

2

6

1

EKS, 750 ppb

60

Absrobance (mAU)

Absrobance (mAU)

3

12 40 4

1 2

3 4

12

4

16 EKS, 7.5 ppb

2

20

0

0 0

5 10 15 Migration Time (min)

20

0

4 8 12 Migration Time (min)

−10 kV due to peak broadening. In the FASI step of EKS, however, the injection amount could be increased to take advantage of the peak sharpening effect of the subsequent tITP step. When the FASI amount was increased, a TE plug longer than the one used in tITP was also needed to ensure sufficient stacking of a longer sample plug. The optimal EKS conditions were FASI of an As sample in 1 mM CHES buffer at −20 kV for 1 min, a 100 mM CHES TE plug injected at 1 psi for 1 min, and then tITP at −20 kV. Using the phosphate run buffer as the LE solution resulted in a simplification of the EKS procedure. During EKS, however, current failures occurred frequently between the FASI and tITP steps since bubbles formed in FASI were introduced into the capillary. This problem was resolved by waiting 30 s after inserting the capillary inlet into a run buffer vial to allow the bubbles to dissolve in the buffer [43]. Thus, the revised optimal EKS conditions were a 100 mM sodium phosphate run buffer, a sample dissolved in 1 mM CHES buffer, FASI at −20 kV for 1 min, a TE plug of 100 mM CHES buffer injected at 1 psi for 1 min, 30 s waiting time, and tITP at −20 kV. The EF and LOD values for a standard sample were 700–8200 and 0.5–1.1 ppb As, respectively (see Table 2). Fig. 4a shows an electropherogram from the EKS of a 750 ppb standard As sample under optimized conditions. Due to these high EF values, the impurity problem resurfaced despite the sample being prepared in a low concentration TE. Fig. 4b compares two EKS electropherograms of 7.5 ppb As samples in 1 mM CHES buffers: one prepared using 99.5% CHES and 97% NaOH and another prepared using 99.99% CHES and 99.5% NaOH (the highest purity commercially available). While the presence of impurity peaks originating from 99.5% CHES and 97% NaOH made peak assignment challenging, reducing impurity peaks through 99.99% CHES and 99.5% NaOH allowed the As peaks to be more reliably assigned.

16

separated with a resolution of 2.1 in the presence of a counter pressure of −0.2 psi (CF-EKS). Note that the resolution values were for non-ideal peaks [45]. Upon increasing the counter pressure to −0.6 psi, the resolution increased to 3.0 as more of the capillary length was used for separation. In contrast, peak heights decreased with a counter pressure due to the reduced net injection velocities of the analytes. Hence, the higher the counter pressure, the lower the amount of injected analyte. This resulted in lower EF values, but improved resolution between peaks. Therefore, the counter pressure optimization had to be carried out to obtain a balance between detection sensitivity and separation quality [44]. In addition, the instability of a μSiL-FC coated fused silica capillary at pH > 10 must also be considered. Although the pH 9.6 of the run buffer and sample buffer was < 10, the local concentration of hydroxide anions could result in higher pH values during a long FASI process in CF-EKS of As anions. In practice, the lifetime of the coated capillary was less than 50 runs when the FASI time exceeded 3 min. Thus, the optimal CF-EKS conditions were similar to those for EKS with the exception of a 3 min FASI step at −20 kV while applying a counter pressure of −0.2 psi. It should be noted that the use of a constant voltage mode for the FASI step in CF-EKS is advantageous when compared to a constant current mode [44]. Fig. 5 shows an electropherogram obtained from CF-EKS of a 560 ppb standard sample. The EFs were 6300–45,000, which are 5–10 times higher than those obtained from EKS, and the LODs of 0.08–0.3 ppb As were obtained (see Table 2). These excellent LOD results obtained using the built-in UV

120

Absrobance (mAU)

CF-EKS, 560 ppb

3.6. Counter flow electrokinetic supercharging (CF-EKS) During the FASI step of EKS, the sample zone migrates forward to the outlet. The amount of sample injected by FASI was limited to secure a sufficient portion of the capillary for the subsequent tITP step. Thus, CF-EKS allows a greater quantity of the sample to be injected, resulting in higher EFs by restricting the movement of the sample zone using a counter flow. In principle, an unlimited quantity of sample can be injected if the sample zone movement is exactly counterbalanced by a counter flow using either pressure or EOF [25,44]. We employed a hydrodynamic counter pressure for this purpose, which was mechanically controlled and therefore easier and more reliable than using an EOF. For a long FASI at −20 kV for 2 min, the DMA and residual carbonate peaks were not baseline separated with a resolution of 0.8 in the absence of a counter pressure (EKS). However, they were baseline

90

3 1 2

60

30

4

0 0

5 10 15 Migration Time (min)

20

Fig. 5. CF-EKS of 560 ppb As in 1 mM CHES buffer. CF-EKS conditions; FASI at −20 kV for 3 min with a counter pressure −0.2 psi, a TE plug of 100 mM CHES buffer injected at 1 psi for 1 min, 30 s waiting, and tITP at −20 kV. The CHES buffer was prepared using high purity chemicals. (99.99% CHES, 99.5% NaOH). Others as in Fig. 2.

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absorbance detector of a commercial CE instrument were more than enough to assess the As species present in drinking water, taking into account the WHO guideline of 10 ppb total As.

As(III) 1.5

3.7. Application to spring water samples

CF-EKS real sample spiked with 15 ppb As

Peak height (mAU)

10

I × V/V0

Prior to analyzing a real sample from a spring, which was designated by the local authorities as non-potable because of As contamination, the CF-EKS conditions needed to be re-optimized due to the presence of complex matrix components in the environmental sample. Thus, we increased the CHES concentration in the sample from 1 to 2 mM, and decreased the FASI time from 3 to 0.5 min to avoid interference from the matrix peaks, particularly on the As(V) peak. The reoptimized conditions were a 100 mM sodium phosphate run buffer, adding 200 μL of 100 mM CHES buffer to a 10 mL sample, FASI at −20 kV with a counter pressure of −0.2 psi for 0.5 min, a TE plug of the 100 mM CHES buffer injected at 5 psi for 0.2 min, 30 s of waiting, and tITP at −20 kV. As shown in Fig. 6, it was possible to clearly assign the As peaks even though the EFs decreased. The LODs were 3.0, 6.0, 2.4, and 9.4 ppb As for As(V), MMA, As(III), and DMA, respectively. The limits of quantification (LOQ, S/N = 10) were 9.9, 20, 7.9, and 31 ppb As for As(V), MMA, As(III), and DMA, respectively. Note that the LOQs for less toxic MMA and DMA were slightly above 10 ppb but the LOQs for more toxic As(III) and As(V) were below 10 ppb. If a coated capillary with a stronger high-pH resistance is available, the sensitivity of CF-EKS can be improved. To quantify the As species present in spring water, aliquots of standard solutions containing 1500 ppb As each of the four As species in deionized water were successively added to a mixture of 10 mL of spring water and 200 μL of 100 mM CHES buffer before CF-EKS analyses were carried out. ICE(V/V0) was plotted as a function of [S]i(VS/ V0) where ICE is the peak height, [S]i the analyte concentration in the standard solution (= 1500 ppb As), V0 the initial sample volume (= 10.2 mL), VS the volume of the added standard solution, and V = V0 + VS. From the x-intercepts of the plots shown in Fig. 7, the concentrations of As(V) and As(III) in the spring water sample were estimated to be 9.2 ± 0.5 and 23.6 ± 0.6 ppb As, respectively, giving a total As content of 32.8 ± 0.8 ppb As. In comparison, the total As levels were also determined by ICP/MS. To 10 mL aliquots of spring water, 0, 1, and 2 mL of standard solutions containing 150 ppb of each of the four As species were added, and the spiked spring water samples were subsequently diluted to 100.0 mL using deionized water. The 75As ICP/ MS signals from the final 100-mL solutions, IICP, were plotted as a

MMA

DMA -23.6

-9.2

0 [S]i × Vs/V0

20

40

Fig. 7. Quantification by standard addition. CF-EKS; 200 μL of 100 mM CHES buffer and 0, 100, 150, 200, or 250 μL of a standard solution containing 1500 ppb As each of the four As species in deionized water were added to 10 mL of spring water. Others as in Fig. 6.

function of the standard concentration in the final solution and the total As level of 35 ± 1 ppb As in the spring water sample was obtained from the x-intercept. This result was in good agreement with the results obtained using our CF-EKS method. 4. Conclusions Among the various on-line sample preconcentration techniques, CFEKS was suitable for analyzing an environmental spring water sample at a sufficiently sensitive level to detect each As species below the WHO guideline of 10 ppb. The application of a counter pressure during the FASI step allowed a greater quantity of sample to be injected, while leaving a sufficient capillary length for the subsequent tITP process. Since this highly sensitive As speciation was achieved using the UV detector of a commercial CE instrument, the proposed method can be considered as readily accessible when compared to current methods that require the use of sophisticated and expensive instruments. Acknowledgment The authors received financial support from the Geo-Advanced Innovative Action Project of the Korea Ministry of Environment (2015000540002).

Carbonate

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