Anal. Chem. 1998, 70, 2666-2675
Use of Triethylenetetraminehexaacetic Acid Combined with Field-Amplified Sample Injection in Speciation Analysis by Capillary Electrophoresis Wuping Liu and Hian Kee Lee*
Department of Chemistry, National University of Singapore, Kent Ridge, Singapore 119260
Simultaneous speciation of lead, mercury, and selenium was carried out by capillary electrophoresis. The method used a polyaminocarboxylic acid, triethylenetetraminehexaacetic acid (TTHA), as an off-column complexing agent to form UV-absorbing complexes with the analytes for direct UV detection. TTHA was also added to the background electrolyte for the on-column complexations of the analytes, as well as for improving resolution and detection. To describe the migration behavior of the complexes, a theoretical model, considerating pH value and the concentrations of TTHA and SDS, was proposed. The parameters in the model were calculated on the basis of the experimental data, by nonlinear regression. The results were in good agreement with those from the literature. The model can be used for the prediction of migration behavior and for the optimization of the separation conditions. Field-amplified stacking injection was performed because the complexes were charged. Up to 1500-fold on-line enrichment and down to sub-nanogramper-milliliter detection limits were obtained for the analytes under the optimal stacking conditions. Finally, the applicability of the method was evaluated on seawater samples. It has been proven that different species of an element have different toxicity to organisms, including human beings.1 Another important feature which distinguishes some toxic species from others is that they are slow to undergo biodegradation, and this contributes to their chronic toxicity. Every year, thousands of tons of metal species are consumed in industry and agriculture for different purposes.1 Some of them are released into the environment and exert toxic influences. To understand the toxicity, bioavailability, bioaccumulation, and transformation of these pollutants, speciation analysis is necessary.2 To date, separation and detection are still the two main challenges in speciation analysis. Among the analytical techniques which are applicable in this area, GC and HPLC are most commonly used. Combined with a variety of methods, such as atomic absorption spectrometry, mass
spectrometry and ultraviolet spectrometry, both techniques have been applied to the speciation analysis of many elements, including tin, lead, and mercury.3-7 To meet the requirements of separation and detection in GC or HPLC, complexation of the analytes, offcolumn or on-column, or even both, is usually needed. While much higher separation efficiency is usually obtained in GC, the availability of a great variety of complexing agents is the advantage of HPLC. Recently, procedures have been carried out to analyze some organometallic compounds by CE.8-10 Complexing agents such as diethyl dithiocarbamate have been used for the preseparation extraction and enrichment of the analytes.10 However, compared to GC and HPLC, the on-line detection sensitivity in CE is relatively lower.11 Several techniques have been reported to be capable of improving the sensitivity in CE.12 Among the developed approaches, field-amplified stacking injection (FASI) is the most promising.13,14 It takes advantage of electrophoretic migration and electroosmosis and has been demonstrated to achieve over 100fold enhancement in detection for charged analytes.15 Through polarity-switching of the electrodes during the stacking process, both positive and negative analytes can be enriched within a single analysis. To perform effective stacking of the charged analytes, it is necessary to prepare the samples in water or in a diluted buffer solution which is of the same composition as the background electrolyte, to generate an amplified field at the injection point of the column. As has been noted,13 when the sample ions are stacked under the amplified field, impurity ions will also be simultaneously concentrated. For real sample analysis, it is important to resolve the impurities from the analytes. Moreover, for the purpose of effective stacking without loss of concentrated
* To whom correspondence should be addressed. E-mail: chmleehk@ nus.edu.sg. Fax: 65-7791691. (1) Craig, P. J. Organometallic Compounds in the Environment-Principles and Reactions; Longman Group Limited: Essex, England, 1986; Chapters 1 and 2. (2) Florence, T. M. Talanta 1982, 29, 345-364.
(3) Liu, Y.; Lopez-Avila, V.; Alcaraz, M.; Beckert, W. F. J. High. Resolut. Chromatogr. 1994, 17, 527-536. (4) Bettmer, J.; Cammann, K.; Robecke, M. J. Chromatogr. A 1993, 654, 177182. (5) Robards, K.; Starr P.; Patsalides, E. Analyst 1991, 116, 1247-1273. (6) Vazquez, M. J.; Carro, A. M.; Lorenzo R. A.; Cela, R. Anal. Chem. 1997, 69, 221-225. (7) Ceulemans, M.; Adams, F. C. J. Anal. At. Spectrom. 1996, 11, 201-206. (8) Han, F.; Fasching, J. L.; Brown, P. R. J. Chromatogr. B 1995, 669, 103112. (9) Ng, C. L.; Lee H. K.; Li, S. F. Y. J. Chromatogr. A 1993, 652, 547-553. (10) Li, K.; Li, S. F. Y.; Lee, H. K. J. Liq. Chromatogr. 1995, 18, 1325-1347. (11) Goodall, D. M.; Williams S. J.; Lloyd, D. K. Trends Anal. Chem. 1991, 10, 272-279. (12) Nielen, M. W. F. Trends Anal. Chem. 1993, 12, 345-356. (13) Burgi D. S.; Chien, R. L. Anal. Chem. 1991, 63, 2042-2047. (14) Jandik, P.; Jones, W. R. J. Chromatogr. 1991, 546, 431-443. (15) Chien, R. L.; Burgi, D. S. Anal. Chem. 1992, 64, 489A-496A.
2666 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
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In this study, the performance and applicability of TTHA in CE were demonstrated by its use in the speciation analysis of lead, mercury, and selenium in different organic and inorganic species. Its effects on separation and detection have been investigated. Moreover, a theoretical model was designed to describe the migration behavior of the analytes, with consideration of the influences of TTHA, as well as pH and SDS. Different parameters in the model were calculated and showed good agreement with literature data. When FASI was used to enhance detectability, factors such as stacking voltage and time and concentrations of TTHA and buffer were evaluated. The effect of FASI was evaluated on the basis of the enhancement in detection that could be obtained. Finally, the applicability of the method was evaluated on real samples. Figure 1. Plot of %TTHA vs pH.
analytes and deterioration in the resolution, it is usually necessary to stop the stacking process when the electric current reaches a certain value, i.e., 95% of that of the pure background electrolyte. Any problem in the stability of analytes in diluted solution, in the monitoring of the current, or in stopping the stacking potential on time will compromise quantitative results.12 These factors may account for the limited number of applications of the FASI technique in real sample analysis.16,17 Triethylenetetraminehexaacetic acid (TTHA) is a polyaminocarboxylic acid with six carboxylic groups;18 its pK3, pK4, and pK5 values are 4.16, 6.16, and 9.40, respectively. TTHA usually forms very stable complexes with most metal ions. In addition, great differences in complexing constants are also found in TTHA complexes. In CE, advantages gained from these properties are manifested as follows: (i) The pKa values of TTHA are in the pH range that is commonly applicable to electrophoresis. (ii) Stable complexes can form at very low concentrations of analytes and TTHA; this is important in FASI, which is applicable to a highly diluted sample mixture. (iii) TTHA4- is the dominant anionic form (>90%) of TTHA in the pH range 7.25-8.0, as depicted in Figure 1 (produced from pKa values and pH). This means that the complexes formed between analytes and TTHA are negatively charged (complexing ratio 1:1 under normal conditions). Consequently, FASI is feasible for their on-column concentration, and electrophoresis can be performed under the normal polarities. (iv) For the speciation analysis of lead and mercury compounds, interferences posed by alkali and alkaline earth metal ions can be alleviated because the complexing constants of these ions with TTHA are very different from those of the complexes of interest. This is important for real sample analyses. Originally, TTHA was used in titration analysis as a masking agent. Recently, its use as a complexing agent has been occasionally reported in the study of rare and transition metals.19,20 However, its application in CE, to our knowledge, has not been described previously. (16) Nielen, M. W. F. J. Chromatogr. 1993, 637, 81-90. (17) Albert, M.; Debusschere, L.; Demesmay, C.; Rocca, J. L. J. Chromatogr. A 1997, 757, 281-289. (18) Cheng, K. L.; Ueno, K.; Imumura, T. Handbook of Organic Analytical Reagents; CRC Press Inc.: Boca Raton, FL, 1982; Chapter 3. (19) Fu, X. T.; Wang, C. M.; Zhang, Y. X. Anal. Chim. Acta 1993, 272, 221225. (20) Paneli, M.; Voulgaropoulos, A. V.; Kalcher, K. Microchim. Acta 1993, 110, 205-215.
THEORY In CE, different theoretical models have been proposed to describe the migration behavior of inorganic metal ions, enantiomers, and other organic compounds.21-23 Vigh et al., in their series reports,22,24,25 studied the correlation between chiral selectivity and peak resolution, and pH and the concentration of different β-cyclodextrins, for the enantiomers of weak bases and weak acids. Unique resolution surfaces were used to describe the different separation types and to optimize the operation conditions. Chen et al.23 proposed a comprehensive theory for an aqueous CE system to describe the migration behavior of analytes involving dynamic equilibria between the analytes and additives. Based on the 1:1 dynamic complexation model, the analyte migration behavior was expressed by an equation comprising the mobilities of the free analyte and the complexes and the equilibria constants for the analyte-additive interactions. For the purpose of explicitly demonstrating the migration behavior of the metallic analytes, a model, based on consideration of the multiple complexing equilibria in the background electrolyte, is introduced here. When a buffer (at a particular pH) is composed of a hexacarboxylic group complexing agent (TTHA), a surfactant agent (SDS), and the analytes, the concentrations of the various complexed species, i.e., the analyte-TTHA, analyte-SDS, and analyte-hydroxo, can be expressed as
[AYb-n] ) βAYn[Ab+][Yn-]
(1)
[Ab+‚‚‚SDS-] ) βAS[Ab+][SDS-]
(2)
[A(OH)mb-m] ) βAOHm[Ab+][OH-]m
(3)
where A represents the analytes, Yn- (n ) 1-6) represents the different anionic forms of TTHA, AYb-n, Ab+‚‚‚SDS-, and A(OH)mb-m are the corresponding analyte-TTHA, analyte-SDS, and ana(21) Yang, Q.; Zhuang, Y.; Smeyers-Verbeke, J.; Massart, D. L. J. Chromatogr. A 1995, 706, 503-515. (22) Rawjee, Y. Y.; Vigh, G. Anal. Chem. 1994, 66, 619-627. (23) Peng, X.; Bowser, M. T.; Britx-Mekibbin, P.; Bebault, G. M.; Morris, J. R.; Chen, D. D. Y. Electrophoresis 1997, 18, 706-716. (24) Rawjee, Y. Y.; Williams, R. L.; Buckingham, L. A.; Vigh, G. J. Chromatogr. A 1994, 688, 273-282. (25) Williams, R. L.; Vigh, G. J. Chromatogr. A 1995, 716, 197-205.
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Table 1. Species of Lead, Mercury, and Selenium in This Study name of species
formula
abbreviation
stock solution concn (µg/mL)
inorganic lead(II) inorganic mercury(II) inorganic selenium(IV) trimethyllead(IV) triethyllead(IV) diphenyllead(IV) phenylmercury(II) phenylselenium(II) diphenylselenium(II)
Pb(NO3)2 HgCl2 Na2SeO3 (CH3)3PbCl (C2H5)3PbCl (C6H5)2PbCl (C6H5)HgCH3COO (C6H5)SeCl (C6H5)2Se
Pb(II) Hg(II) Se(IV) TMLC TELC DPLC PMA PSC DPS
358 442 1000 5080 5900 2550 1000 2800 1000
lytes-hydroxo complexes, and βAYn, βAS, and βAOHm are the stability constants, respectively. TTHA is a polycarboxylic acid, and its protonation in aqueous solutions has to be taken into consideration. This is defined as δyn ) [Yn-]/Cy, or [Yn-] ) Cyδyn (Cy is the total concentration of TTHA in the background electrolyte), which indicates the molar fractions of TTHA in different anionic forms which are capable of reacting with the analytes. Depending on the conditions, both mononuclear and binuclear TTHA complexes can be formed for some metal ions.26 However, under conditions when the amount of TTHA is in great excess of that of the analytes, just as was used in this work, only 1:1 complexes need to be considered. Moreover, when the complexing agent is added in an equilibrium amount or in excess, the free analyte concentration will be so low that no polynuclear hydroxo complexes will be present.27 Consequently, only mononuclear hydroxo complexes are considered. Protonation, or the influence of the hydroxide ion, on the complexes was neglected for the sake of simplicity, because the migration behavior of all the analyte-TTHA complexes was studied under weakly acidic to basic conditions. Also, the influence of the buffer solution was not taken into account here, based on the common extrapolation that its effect on both migration and detection is insignificant, provided that it is being used at a constant and rather high concentration that supplies enough buffer capacity. The total concentration of each analyte, CA, is the sum of various chemical species present in the buffer: b+
CA ) [A ] +
∑[AY
b-n
] + [A ‚‚‚SDS ] +
∑ ∑(β
n n AY δy )
+ βAS[Ab+][SDS-] +
b+
∑
[A ]
(βAOHm[OH-]m)
(4)
Therefore, the molar fraction of the analytes in the free and various complexed species can be expressed by combining eqs (26) Harju, R. Anal. Chim. Acta 1970, 50, 475-489. (27) Ringbom, A. Complexation in Analytical Chemistry; Interscience Publishers: New York, NY, 1963; Part II and Appendix.
2668 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
+ βAS[SDS-] +
n n AY δy )
∑(β
m - m AOH [OH ] )]
βAS[SDS-] +
n n AY δy )+
∑(β
m - m AOH [OH ] )]
δAS )
∑(β
[Ab+‚‚‚SDS-]/CA ) βAS[SDS-]/[1 + Cy -
βAS[SDS ] +
(5)
∑(β
δAYn ) [AYb-n]/CA ) βAYnδynCy/[1 + Cy
∑
n n AY δy )
(6)
+
(βAOHm[OH-]m)]
(7)
δAOHm) [A(OH)mb-m]/CA
∑(β δ [SDS ] + ∑(β
) βAOHm[OH-]m/[1 + Cy
n n AY y )
-
βAS
+
m - m AOH [OH ] )]
(8)
The effective electrophoretic mobility, µeff, of each analyte is equal to the weighted average of the electrophoretic mobilities of the different free species of the analytes:
∑µ ∑δ ) {µ + C ∑µ ∑(β ∑µ ∑(β [OH n AY
µeff ) µ0 δA + 0
y
n AY
+ µASδAS +
n n AY δy )
AY
m AOH
∑µ
∑δ
m AOH
m AOH
+ µASβAS[SDS-] +
- m
∑(β
] )}/{1 + Cy
βAS[SDS-] +
[A(OH)mb-m]
) [Ab+] + [Ab+]Cy
∑(β
δA ) [Ab+]/CA ) 1/[1 + Cy
m AOH
-
b+
1-4, as
∑(β
n n AY δy )
+
m - m AOH [OH ] )}
(9)
where µ0 is the electrophoretic mobility of the free analyte, and µAY, µAS, and µAOH are the electrophoretic mobilities of the various complexed species. This equation is similar in form to the mobility expression derived from the dynamic complexation model23 and presents a typical example of multiple equilibria in CE. In our situation, eq 9 gives a general expression of the analyte migration behavior for speciation analysis by CE. It indicates that the mobility of the analytes is related to the different β values and the condition-dependent parameters, i.e., pH of the buffer, and concentrations of the complexing agent and SDS. Changes in these factors will result in variation in µeff.
EXPERIMENTAL SECTION Instrumentation. Experiments were performed on a PRINCE (Emmen, The Netherlands) CE system. A Lambda 1000 (Bischoff, Leonberg, Germany) UV/visible detector was used with the wavelength set at 200 nm for detection. Data acquisition was carried out with a Shimadzu C-R6A Chromatopac (Tokyo, Japan). The separation capillary was 64.5 cm long (52 cm effective length) and 50 µm i.d. Hydrodynamic injection of the samples was conducted by pressure (50 mbar for 0.1 min) at the anodic end of the capillary. Electrophoresis was carried out at +20 kV voltage. When FASI was used for on-line sample stacking, a small plug of water was hydrodynamically injected (50 mbar, 0.1 min) just before the polarity-switching field-amplified injection of the solutes (+10 kV for 2.0 min, and -10 kV until 95% of the current of the pure background electrolyte was reached). Nonlinear regressions were performed in Microsoft Excel 97 using a Compaq personal computer. Reagents and Standard Solutions. Three elements, lead, mercury, and selenium, were studied as their organic species (Aldrich Co., Milwaukee, WI) and inorganic species (J. T. Baker, Phillipsburg, NJ). The stock solutions were prepared in water (inorganic species) or in HPLC-grade methanol (Fisher Scientific, Fair Lawn, NJ) (organic species). The names, formulas, abbreviations of the species, and the concentrations of their stock solutions are listed in Table 1. For convenience, the abbreviations are used to represent the corresponding analyte-TTHA complexes in the discussion below. Aqueous solutions of 185 µg/mL Na(I), 162 µg/mL Ca(II), and 50 µg/mL Mg(II) were prepared as the major interfering ions in real sample analysis. Aqueous stock solutions of 50 mmol/L (mM) TTHA (Merck Chemicals, Darmstadt, Germany), 200 mM sodium dodecyl sulfate (SDS) (Fluka, Buchs, Switzerland), and 100 mM sodium tetraborate (Fluka) were also used. To prepare the TTHA stock solution, a specific amount of TTHA (powder) was first wetted with water, and then 1.0 M NaOH solution was added dropwise until almost all the powder was dissolved; finally, the mixture was made up to volume with water. The sodium tetraborate solution was mixed with 500 mM phosphoric acid [diluted from 85% phosphoric acid (Carlo Erba, Italy)], to the needed pH; this buffer solution was then diluted with water, according to the specific conditions, and used in the background electrolyte or sample mixture. Deionized water used throughout the experiment was produced on a Milli-Q system (Millipore, Bedford, MA). Sample Preparation and Spiking of Seawater in FASI. Offcolumn complexation was carried out by mixing the stock solutions of the analytes (stored under ∼4 °C) with the buffer and TTHA solutions in a glass sample vial; the sample volume was made up to 200 µL with water for CE analysis. In the case of on-column complexation, procedures similar to the one described above were adopted for sample preparation, except with the absence of TTHA; complexation occurred during CE separation within the capillary, where the analytes complexed with TTHA in the background electrolyte. For real sample analysis, 100 µL of tap water or seawater was used instead of the stock solutions. In the study of the accuracy and precision of the method, the tap water or seawater was spiked with known amounts of the analytes. In hydrodynamic injection, the buffer and TTHA solutions for preparing the sample mixtures were at the same concentrations
Figure 2. Plot of electrophoretic mobility (µeff) vs pH. Column, fusedsilica capillary (64.5 cm × 50 µm i.d.) with 52-cm length for separation; injection, 50 mbar × 0.1 min; voltage, 20 kV; detection, 200 nm; background electrolyte, 40 mM, pH 6-8.5, NaH2PO4-Na2B4O7 buffer; sample mixture, background electrolyte and analytes.
as those used in the background electrolyte, while in FASI, they were diluted before use. Surface seawater samples (1.5 L each) were collected from the south coast of Singapore (20 m from the beach). After being kept at room temperature for 12 h, the samples were filtered twice with no. 4 sintered funnels to remove particulates before use. Calculation. Electrophoretic mobility (µeff) is calculated using eq 10:
µeff )
( )
LtLd 1 1 V t t0
(10)
where Lt and Ld are the total and effective lengths of the capillary, respectively, V is the applied voltage (20 kV), t the migration time of the analyte, and t0 the migration time of methanol, which is used as the electroosmotic flow (EOF) marker. The corrected peak area was used to evaluate the effect of different parameters on the detection of the analytes and detection enhancement by stacking injection.17 RESULTS AND DISCUSSION Effect of pH. pH is a very important factor in CE. Its influence on the migrations and separations has been extensively studied.25 When borate buffer is used, the concentration of borate ion will also change accordingly, as will the ionic strength of the solution if balancing ions were not added.22 More important, the variation in acidity of the electrolyte will affect the dissociation, the existence of TTHA, and its complexing ability with analytes. To identify the net influence of pH on the migration behavior of the analytes, measurements were first conducted under weakly acidic to basic conditions, pH ∼6.0-8.5, with the absence of TTHA and SDS. Shown in Figure 2 is the plot of electrophoretic mobility vs pH for some analytes. The decrease in electrophoretic mobility is observed for triethyllead (TELC) and phenylmercury (PMA). While the electrophoretic mobility of phenylselenium (PSC) increases very slightly, Se(IV) has a steadily increasing trend in Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
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its electrophoretic mobility. TMLC migrated faster than the EOF marker (it exhibited a positive electrophoretic mobility) at lower pHs (
