ppt Levels with Capillary

IC is capable of achieving low-parts-per-trillion (ppt) detection limits; however, ..... Quantitative Analysis of Anions in the Range of 0.5−5 ppb (...
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Anal. Chem. 1998, 70, 2261-2267

Quantitative Analysis of Anions at ppb/ppt Levels with Capillary Electrophoresis and Conductivity Detection: Enhancement of System Linearity and Precision Using an Internal Standard Carsten Haber* and Richard J. VanSaun

Orion Research, 500 Cummings Center, Beverly, Massachusetts 01915 William R. Jones

Thermo Bioanalysis, 8 East Forge Parkway, Franklin, Massachusetts 02038

Capillary electrophoresis with conductivity detection is evaluated for quantitative analysis of anions at low- to subppb concentration levels in the presence and absence of a conductive sample matrix composed of 2 ppm ammonia and 50 ppb hydrazine. The low-level sensitivity is extended by a transient isotachophoretic stacking procedure. The linear range of the CE system and conductivity detector is graphically evaluated on the basis of absolute and corrected (normalized) chloride and sulfate peak profiles using an ASTM linearity criterion. The influence of random contamination bias from ubiquitous entities of nonsample chloride and sulfate levels introduced by liquid handling, laboratory atmosphere, and bulk chemical residues is quantitatively compared against an internal (contamination) reference ion. Unlike suppressed ion chromatography, which is to date the established separation technique for quantitative trace-level ion analysis, capillary electrophoresis (CE) permits direct electrostacking of analyte ions from a dilute solution to form a narrow and concentrated sample plug in the separation compartment. This fundamental electrophoretic phenomenon is based on the Kohlrausch regulating function1 and automatically predetermines the degree of analyte stacking as a function of its mobility/concentration ratio versus the background electrolyte.2 When optimized, the process of electrokinetic loading (stacking) sample solution can produce sensitive and highly resolved separations.3 In recent years, considerable efforts have been made in combining elements of isotachophoresis (ITP) with CE for the purpose of sensitivity enhancement. The process of confining a liquid sample plug into a transient ITP (t-ITP) zone pattern 4-14 (1) Kohlrausch, F. Ann. Phys. Chem. 1897, 62, 209-39. (2) Klepa´rnı´k, K.; Bocˇek, P. J. Chromatogr. 1991, 569, 3-42. (3) Chien, R. L.; Burgi, D. S. Anal. Chem. 1992, 64, 489A-96A. (4) Beckers, J. L.; Everaerts, F. M. J. Chromatogr. 1990, 508, 19-26. (5) Verheggen, Th. P. E. M.; Schoots, A. C.; Everaerts, F. M. J. Chromatogr. 1990, 503, 245-55. (6) Dolnik, V.; Cobb, K. A.; Novotny, M. J. Microcolumn Sep. 1990, 2, 12731. S0003-2700(97)01210-9 CCC: $15.00 Published on Web 04/24/1998

© 1998 American Chemical Society

can be a relatively fast and simple procedure to boost the total system sensitivity by more than 100 times. The procedure can be carried out in conventional CE instrumentation. Anions at parts-per-billion (ppb) concentration levels are monitored on a frequent basis in ultrapure water utilized for steam generation in fossil and nuclear power plants. It has been recognized that finite amounts of chloride and sulfate, among other factors, can contribute to equipment damage through corrosion or deposition. Chloride can be a significant contributor to corrosion in boilers and turbines. Guidelines by the Electric Power Research Institute15 suggest that chloride concentrations be maintained below 3 ppb in the steam of reheater units and below 6 ppb in the superheated steam of nonreheat units.16 Chloride intrusions commonly result from condenser leaks and sometimes from condensate polishing or makeup demineralizers. The resultant ingress of chloride can ultimately reach the turbine as a result of mechanical or vaporous carry-over, with potential damage to blade materials in the turbine generator. The determination of sulfate is utilized in the assessment of steam purity, condenser inleakage, condensate polisher, and makeup demineralizer performance. To protect turbine and turbine components from corrosion effects, sulfate concentration in the steam should be 3 ppb or less.16 (7) Gebauer, P.; Thormann, W.; Bocˇek, P. J. Chromatogr. 1992, 608, 47-57. (8) Schwer, C.; Gasˇ, B.; Lottspeich, F.; Kenndler, E. Anal. Chem. 1993, 65, 2108-15. (9) Foret, F.; Szo ¨ko¨, E.; Karger, B. L. Electrophoresis 1993, 14, 417-28. (10) van der Vlis, E.; Mazereeuw, M.; Tjaden, U. R.; Irth, H.; van der Greef, J. J. Chromatogr. 1994, 687, 333-41. (11) Janini, G. M.; Muschik, G. M.; Issaq, H. J. J. Capillary Electrophor. 1994, 1 (2), 116-20. (12) Krˇiva´nkova´, L.; Gebauer, P., Bocˇek, P. J. Chromatogr. A 1995, 716, 3548. (13) Witte, D. T.; Nagard, S.; Larsson, M. J. Chromatogr. A 1994, 687, 155-66. (14) Reinhoud, N. J.; Tjaden, U. R.; van der Greef, J. J. Chromatogr. A 1993, 653, 303-12. (15) Guideline Manual on Instrumentation and Control for Fossil Plant Cycle Chemistry, Final Report; Electric Power Research Institute: Palo Alto, CA, 1987. (16) Interim Consensus Guidelines on Fossil Plant Cycle Chemistry; Electric Power Research Institute: Palo Alto, CA, 1986.

Analytical Chemistry, Vol. 70, No. 11, June 1, 1998 2261

In nuclear power plants the required detectabilities generally extend into the sub-ppb levels (0.1-0.2 ppb chloride and 0.2-0.5 ppb sulfate). The low-level analysis is further complicated by the presence of numerous feedwater additives for the control of pH and dissolved oxygen, e.g., ammonia, morpholine, and hydrazine/ carbohydrazide. Other anions (various organic acids, nitrate, fluoride, phosphate, silica, sulfite) are monitored depending on the type of facility, feedwater/chemical deaeration additives, and corresponding breakdown products. As of today, the majority of the existing power industrial QC/ QA applications for anions are based on ion chromatography (IC) with suppressed conductivity detection. IC is capable of achieving low-parts-per-trillion (ppt) detection limits; however, it is necessary to concentrate sufficient mass of analyte into a narrow starting band of the IC column (typically 1% of the column volume). This procedure is typically carried out off-line via trap-release device methods and requires extra components (preconcentration pump, automatic valve, preconcentration column, etc.). Depending on the extracted analyte volume, the preconcentration process can be very time-consuming (up to 30 min for the analysis of ppt levels) while recovery and contamination bias may be a problem. Conductivity detection for CE recently became commercially available.17,18 This detection mode is based on mobility differences between eluting zones and does not suffer from the LambertBeer limitation.19 The conductivity response is ∼10-fold more sensitive than indirect UV detection techniques and found to be linear over more than 3 orders of magnitude. Low mobility electrolytes of high ionic strength allow for more robust separations, while peak asymmetries, frequently encountered due to mobility “mismatch” in indirect UV, are less pronounced. Although CE is proven as a rapid, highly efficient, and sensitive analysis technique, only a fraction of the scientific and technical contributions are explicitely dedicated to quantitative low-level ion analysis applicable to the industrial QC/QA environment.20-24 In the present work, we describe a rapid field-amplified/t-ITP sample injection procedure in combination with conductivity detection which is potentially applicable to the QC/QA requirements in the power industry. The instrumental procedure is tested in particular for quantitative low-level analysis of chloride and sulfate using an internal standard and external calibration. The impact of contamination bias and matrix effects on the total system linearity is evaluated on the basis of an ASTM linearity criterion. EXPERIMENTAL SECTION Instrumentation. All components of the CE system except for data acquisition are available from Thermo BioAnalysis (Franklin, MA; formerly Thermo Capillary Electrophoresis; http: (17) Jones, W. R.; Soglia, J.; McGlynn, M.; Haber, C.; Reineck, J.; Krstanovic, C. Am. Lab. 1996, 5, 25-33. (18) Haber, C.; Jones, W. R.; Soglia, J.; Surve, M. A.; McGlynn, M.; Caplan, A. Reineck, J. R.; Krstanovic, C. J. Capillary Electrophor. 1996, 3 (1), 1-11. (19) Pentoney, S. L.; Sweedler, J. V. In Handbook of Capillary Electrophoresis, 2nd ed.; Landers, J. P., Ed.; CRC Press: Boca Raton, FL, 1997; Chapter 12. (20) Jones, W. R.; Jandik, P. J. Chromatogr. 1991, 546, 431-43. (21) Bondoux, G., Jandik, P. Jones, W. R. J. Chromatogr. 1992, 602, 79-88. (22) Carpio, R. A.; Mariscal, R. Anal. Chem. 1992, 64, 2123-9. (23) Boden, J.; Ba¨chmann, K.; Kotz L.; Fabry, L.; Pahlke, S. J. Chromatogr. 1995, 696, 321-32. (24) Ehmann, Th.; Ba¨chmann, K.; Fabry, L.; Ru ¨ fer, H.; Pahlke, S.; Kotz, L. Chromatographia 1997, 45, 301-11.

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\\www.ThermoCE.com). The Crystal 310 autosampler is the modular base unit and serves as the CE front end throughout all the experiments. The autosampler is combined with the Crystal CE 1000 conductivity detector. A description of the detector hardware, conductivity cell, capillary (ConCap), and conductivity sensor (ConTip) has been given elsewhere.17, 18 The experiments are carried out using uncoated fused-silica capillary of 50-µm i.d. and 60-cm length (ConCap) fitted into both modular units as already decribed. For all analysis cycles, oven and conductivity cell temperature are thermostated at 35 °C. Data Acquisition. The data acquisition, peak identification, integration, and calibrations are carried out with the EZChromJ chromatography data system (Scientific Software, Inc., San Ramon, CA). Data are acquired with the SS 420 ADC card at a sampling frequency of 12.5 Hz. Volumetric Equipment, Vials, and Fluid Handling. Volumetric equipment used for the preparation/dilution of ppb/ppt calibration standards and samples is made from polypropylene or poly(methylpentene) (Cole-Parmer, Vernon Hills, IL). Plastic material is advantageous, since it has generally little extractable material from the wall. All volumetric flasks, containers, and sample vials including snap-top starburst septa are soaked for a minimum overnight period in 18-MΩ water. Disposable pipetting material is consequently cleaned with 18-MΩ water and prerinsed with the corresponding analyte/reagent before usage. Sample solutions and calibration standards containing ppb/ppt amounts of ions are prepared and handled in large volumes in order to minimize the relative influence of point, surface, and aerosol contamination ingress during handling and filling procedures. Autosampler Vials. Autosampler vials for the Crystal 310 are available in glass and plastic. Both vial types have identical external dimensions and can be sealed with a snap-top starburst septum, which is important to cut off the influence of atmospheric aerosol ingress. The plastic vials (J.G. Finneran Associates, Inc., Vineland, NJ), which hold an ∼2-mL volume, are preferentially used for the leading electrolyte and sample solutions. The glass vials hold a larger amount of liquid (4.5 mL) and are conveniently used to hold the electrolyte. Larger electrolyte volumes are generally more favorable against electrolytic degradation under the influence of the high voltage. Electrolytes and Solutions. All solutions are consequently prepared with 18-MΩ water (Milli-Q UV PlusJ, Millipore Corp., Bedford, MA). Running Electrolyte. The running electrolyte used throughout the experiments consists of 50 mM boric acid (99%; SigmaUltra, Sigma Chemical Corp., St. Louis, MO), 20 mM LiOH (99.95%; Aldrich Chemical Co., Milwaukee, WI), 0.1 mM TTAOH, and 0.75% Triton X-100 at a natural pH of 8.9. Some earlier experiments were carried out with an equivalently performing mixture of 50 mM (2-(N-cyclohexylamino)ethanesulfonic acid (CHES, minimum 99%, Sigma Chemical Corp.), 20 mM LiOH (99.95%, Aldrich Chemical Co.), and 0.03% Triton X-100 at a natural pH of 9.2. To reverse the electroosmotic flow, a separate capillary flush with 1 mM (cetyltrimethylammonium bromide (CTAB; Sigma Chemical Corp.) solution is required. All electrolytes are degassed and microfiltered (MilliCupJ filter unit 0.45 µm, Millipore Corp., Bedford, MA).

Leading Electrolyte. The leading electrolyte is 10 mM LiOH (99.95%, Aldrich Chemical Co.), prepared daily. Flow Modifier. The flow modifier tetradecyltrimethylammonium bromide (TTAB 99%; Sigma Chemical Corp.) is converted from the bromide form to the hydroxide form using Dowex I nuclear grade ion-exchange resin (hydroxide form, dry mesh: 2050; capacity 1.1 mequiv/mL; Sigma Chemical Corp.). Triton X-100. Commercial Triton X-100 contains even in its purest available form (Scintillation Grade, Acros Chemicals) residual levels of chloride, sulfate, and phosphate. When used in the running electrolyte for trace-level applications, Triton X-100 is purified by ion-exchange using Dowex I nuclear grade ionexchange resin (see above). Terminating Electrolyte/Internal Standard. To simplify the sample preparation, terminator and internal standard are combined in a single solution (2000 ppm sodium octanesulfonate, purified, VHG Labs, Manchester, NH; 15 ppm sodium-tungstate, Fluka Chemical Corp., Ronkonkoma, NY). The sample preparation requires a one-step 1:1000 dilution of terminator/internal standard for each sample and calibration standard. Matrix Components. Ammonia and hydrazine (Fisher Scientific Co., Pittsburgh, PA) are used as matrix components and added to a series of calibration standards. RESULTS AND DISCUSSION ITP Stacking Conditions. In a transient isotachophoretic stacking mode, the sample is placed (“sandwiched”) between a leading and terminating ion. The t-ITP step has to be optimized for each individual electrolyte system. The type of running electrolytes used throughout this work have lower mobilites as compared to the analyte ions and could potentially take over the function of a terminator. We found, however, that the sensitivity still improved when a separate terminator of lower mobility (octanesulfonate) was added to the sample. The optimum concentration was experimentally determined to be ∼2 ppm. Lower concentrations as well as concentrations exceeding 5 ppm were found to deteriorate the results. The leading electrolyte (0.01 M LiOH) is hydrodynamically injected prior to the sample using a pressure pulse of 40 mbar and 24-s duration. The zone length thus corresponds to ∼12.5 mm (2.1% of total capillary volume). We found that small differences ((5 mbar) in the injection of the leading ion did not change the sensitivity considerably. The system performance was found to be more susceptible to changes in concentration and decreases considerably at higher (0.1 M LiOH) or lower concentration (1 mM LiOH). No t-ITP stacking effect was observed when the leading electrolyte was mixed with the background electrolyte before injection. The sample, which contains the terminating ion (2 ppm octanesulfonate) and internal standard (15 ppb tungstate) is loaded electrokinetically up to -3 kV at 45 s. Longer injection times or higher injection voltages distort the peak profiles and therefore decrease the overall performance, probably due to the differences in EOF between the sample and the carrier electrolyte and the resulting buildup of electroosmotic pressure which induces peak broadening of the sample zone.25 For higher concentrated samples, the loading time is reduced appropriately in order to avoid overloading effects. (25) Chien, R. L.; Helmer, J. C. Anal. Chem. 1991, 63, 1354-61.

Figure 1. Electrokinetic injection of a 1 ppb sample (A) and under t-ITP stacking conditions (B). Sample chloride and sulfate, 1 ppb each; sample preparation, 30 ppb tungstate (internal standard), 5 ppm octanesulfonate (terminator) added to sample solution; electrolyte, 50 mM CHES, 20 mM LiOH, 0.03% (w/w) Triton X-100 at pH 9.2; capillary, 50 µm × 60 cm; separation voltage, -25 kV; injection (A) -5 kV × 24 s, no preinjection of leading electrolyte (ITP criterion not met); (B) - 5 kV × 24 s, preinjection of leading electrolyte, 40 mbar × 18 s.

The system performance is also effected by carry-over effects from the highly conductive LiOH. To maintain the sample integrity, we therefore placed an intermediate 18-MΩ water/ “cleaning dip” between leading electrolyte and the sample in order to strip off the residual highly conductive fluid from capillary end and Pt wire. The efficiency of the t-ITP stacking procedure is illustrated in Figure 1. The upper trace (A) is acquired without preinjection of LiOH, whereas the full ITP stacking criterion is met in the lower trace (B). A similar result is also found, when the terminating ion (octanesulfonate) is not added to the sample. Analysis Method. The system reproducibility is maximized by operating the electrophoretic separation under isothermal (35 °C) and constant-current conditions. To maintain uniform surface conditions and ensure the best reproducibility of the ζ-potential/ EOF conditions in the fused-silica capillary, the capillary is stripped with 0.1 N NaOH and subsequently rinsed with 18-MΩ water after every run. Vial arrangement, filling mode, liquid level, vial material, and capping are tested for cross-contamination effects. All solutions are carefully handled with clean volumetric material (autosampler vials, caps; see above) and consequent wearing of fingercots by the operator. Clean (plastic) autosampler vials are repetitively rinsed with 18-MΩ water and sample solution from a 16-oz dropping bottle (Cole Parmer, Vernon Hills, IL). The vials are then refilled with the respective sample/calibration standards, capped, and used for a single injection. Internal Standard. Peak area normalization with an internal standard26-28 can compensate for bias from electrokinetic injection29 and flow variations. Analytical Chemistry, Vol. 70, No. 11, June 1, 1998

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Sodium tungstate is used as a nonubiquitous ion throughout the analysis as an internal standard. The concentration is set at 15 ppb for calibrations ranging from 500 ppt to 5 ppb and 30 ppb for calibrations ranging from 1 to 20 ppb of sample ion, respectively. Detector Linearity. Accurate quantitative analysis requires rigorous detector linearity. In the majority of the scientific literature, detector linearity is preferentially displayed by fitting a least-squares line through the obtained data. However, it is wellknown that nonlinear response curves can lead to r2 values of 0.9 and higher.30 Dorschel et al.31 indicated that the process of fitting a straight line tends to smooth the data and obscure any evidence of nonlinearity, especially for the lowest concentration standards.32 The linearity of a detector can be more accurately assessed in dividing the detector response (R) by the corresponding concentration (C) and observing whether the values thus obtained are in fact constant to within some defined limit. The ASTM practices for evaluating GC detectors employ this definition and specify a tolerance of (5%.33 A graphical representation contains the R/C ratios for a selected concentration range which are, under ideal circumstances, confined by the (5% linearity criterion. This socalled linearity plot provides the most rigorous and useful evaluation of the suitability of a linear model for the detector response and will be applied as a guideline throughout this work. System Linearity. Adequate detection linearity is a fundamental requirement for precise quantitative analysis. In practice, however, it becomes more logical and useful to evaluate the linearity of the whole separation system including the detector, which represents as a whole entity the effective tool in determining the unknown concentration of individual sample components. Sampling effects (from, for example, injection fluctuations and electrostacking bias) and CE separation characteristics (e.g., nonGaussian peak shape and nonuniformity of the electroosmotic flow) can bias and impair the true detector linearity, although the detector itself might prove linear over several orders of magnitude. An evaluation of the system linearity under t-ITP stacking conditions for chloride is shown in the Figure 2. The linear regression calculated on the absolute areas of chloride in the range 2-20 ppb seems to indicate adequate system linearity (r2 ) 0.986; see Table 1) at the first glance. The corresponding linearity plot, however, reveals that true system linearity for chloride according to the (5% guideline is strictly limited to the range between 10 and 20 ppb (Figure 2A). To qualitatively visualize random contamination bias (from sample handling, atmospheric ingress, etc.), we chose molybdate as a nonubiquitous contamination reference ion for direct in-sample comparison against chloride. A similar regression calculated on (26) Huang, X.; Luckey, J. A.; Gordon, M. J.; Zare, R. N. Anal. Chem. 1989, 61, 766-70. (27) Dose, E. V.; Guiochon, G. A. Anal. Chem. 1991, 63, 1154-8. (28) Qi, S.; Huang, A.; Sun, Y. Anal. Chem. 1996, 68, 1342-46. (29) Huang, X.; Gordon, M. J.; Zare, R. N. Anal. Chem. 1988, 60, 375-7. (30) Analytical Methods Committee, Royal Society of Chemistry Analyst 1988, 113, 1469-71. (31) Dorschel, C. A.; Ekmanis, J. L.; Oberholtzer, J. E.; Warren, F. V.; Bidlingmeyer, B. A. Anal. Chem. 1989, 61, 951A-68A. (32) Colin, H.; Guiochon, G.; Martin, M. In Practice of High Performance Liquid Chromatography; Engelhard, H., Ed.; Springer-Verlag: Berlin, 1986; p 31. (33) Annual Book of ASTM Standards; American Society for Testing and Materials: Philadelphia, 1988; pp 149-58, 178-83, 235-45, 289-99, 3448.

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Table 1. Linear Regression Statistics for Chloride and Molybdate

chloride (absolute area) chloride (corrected area) molybdate (absolute area) molybdate (corrected (area)

r2 value

constant (y-intercept)

std error of coeff

0.9860 0.9939 0.9857 0.9997

4.0469 -0.0053 2.5407 -0.008

0.2228 0.0127 0.089 0.001

Figure 2. Linearity plot of chloride [absolute areas (A); corrected areas (B)]. Each point average of three consecutive runs (error bars, RSD of area counts). Calibration standards (range) chloride (2-20 ppb), sulfate (4-40 ppb), molybdate (5-50 ppb); sample preparation, internal standard (tungstate, 10 ppb), terminator (propionate, 1 ppm) added to each calibration standard; electrolyte, 50 mM CHES, 20 mM LiOH, 0.03% (w/w) Triton X-100; capillary, 50 µm × 60 cm, preflushed with 1 mM CTAB for 24 s at 2000 mbar; preinjection of 0.01 M LiOH, 50 mbar x 18 s; injection, -5 kV × 24 s; separation voltage/current, -25 kV/-11.1 µA.

the molybdate peak area over a range of 5-50 ppb (r2 ) 0.986; see Table 1) and corresponding linearity plot (not shown) confirms the same trend as chloride. When judged by the linearity plot on the absolute areas, the system obviously does not behave truly linear at the lower concentration end. The apparently larger peak area at lower sample concentrations for chloride is a combination of biases due to electrostacking and contamination effects. The two contributions can be separated in dividing (normalizing) the corresponding peak areas of chloride by the area of the internal standard. This operation practically

Figure 3. Trace analysis of anions in a matrix of 1 ppm ammonia and 50 ppb hydrazine. Sample A, chloride (1), sulfate (2), molybdate (3), 1 ppb each; sample B, chloride (1), sulfate (2), molybdate (3), 20 ppb each; sample preparation, internal standard (tungstate, 30 ppb), terminator (octanesulfonate, 5 ppm) added to each standard; electrolyte, 50 mM CHES, 20 mM LiOH, 0.03% (w/w) Triton X-100 at pH 9.2; capillary, 50 µm × 60 cm, preflushed with 1 mM CTAB for 24 s at 2000 mbar; preinjection of 0.01 M LiOH, 40 mbar x 18 s; injection: -5 kV x 24 s; separation at -10 µA.

eliminates the electrokinetic bias for chloride and equally improves the regression statistics of a least-squares fit for the area count vs concentration (r2 ) 0.994; see Table 1). The linear range for chloride is now extended toward lower concentrations except for the 2 ppb data point, which apparently is biased by contamination (Figure 2B). The presented data illustrate that peak area correction extends the linear range and enhances system precision. Internal standard and area correction are therefore categorically applied for trace ion analysis using electrokinetic loading and t-ITP stacking conditions. System Linearity in the Presence of a Conductive Matrix. In many cases, sample ions must be analyzed in the presence of a conductive matrix. A fundamental drawback of electrokinetic injection techniques in CE is the fact that the system sensitivity generally suffers with increasing concentration of nonsample () matrix) ions. Figure 3 shows an ion electropherogram of a sample containing chloride, sulfate, and molybdate at the low (1 ppb each, trace A) and high end (20 ppb each, trace B) of the calibration, each with 30 ppb tungstate (internal standard) in a matrix of 1 ppm ammonia and 50 ppb hydrazine injected electrokinetically under standard t-ITP stacking conditions. Ammonia and hydrazine are widely used in the electric power industry and added to the feedwater for pH adjustment and oxygen control. Typical matrix compositions may consist of up to 1 ppm ammonia and 50 ppb hydrazine. Both matrix constituents increase the total conductivity of the sample but do not directly interfere with the separation. The corresponding linearity plot of the area-corrected sulfate signal (Figure 4) indicates that the 1 ppb sample is, similar to chloride (Figure 2B), slightly biased by contamination. However, the improved linear regression statistics (Sulfate: r2 ) 0.9997, constant, -0.004. Chloride: r2 ) 0.9997; constant, 0.008) suggests that the presence of a finite and constant conductive samplematrix obviously improves the electrokinetic loading process.

Figure 4. Linearity plot of sulfate in a matrix of 1 ppm ammonia and 50 ppb hydrazine (corrected areas). Each calibration point comprises the average of three consecutive runs (error bars, RSD of area counts). Sample preparation, internal standard (tungstate, 30 ppb), terminator (octanesulfonate, 5 ppm) added to each calibration standard; electrolyte, 50 mM CHES, 20 mM LiOH, 0.03% (w/w) Triton X-100, pH 9.2; capillary, 50 µm × 60 cm, preflushed with 1 mM CTAB for 24 s at 2000 mbar; preinjection of 0.01 M LiOH, 50 mbar × 18 s; injection, -5 kV × 24 s; separation at -10 µA.

Quantitative Analysis of Anions in the Presence of a Conductive Sample Matrix (1 ppm Ammonia, 50 ppb Hydrazine). In the following experiment, we spiked several solutions containing 50 ppb hydrazine, 1 ppm ammonia, and the previously indicated amounts of internal standard and terminator with various known quantities of sulfate and molybdate. The recoveries of both ions are calculated on the basis of a peak areacorrected five-point linear regression. The results are listed in Table 2 and indicate that the analysis method produces acceptable results for the quantification of low-ppb anions. A short statistical survey of the sample recoveries yields an average relative error of 7.8% for chloride and 8.7% for molybdate, respectively. This finding is somewhat of a paradox, since molybdate as a nonubiquitous contamination reference ion should account for a smaller relative error than sulfate, which is more bias prone due to the influence of random contamination. There is obviously a finite sulfate offset in all samples since the found concentration levels including the blank (sample 4) are always higher than the added amount. However, this sulfate offset has obviously less impact on the precision of the corresponding peak area recoveries than random profile fluctuations of molybdate. Changing Matrix Composition. We investigated the impact of changing matrix conductivity on the peak area reproducibility of the analyte components. A 10 ppb sample was injected under t-ITP stacking conditions by increasing the matrix composition from 0 to 2 ppm ammonia and 0 to 100 ppb hydrazine, respectively. The absolute peak areas of chloride (Figure 5A) reflect the trend of decreasing system sensitivity with increasing concentration of the matrix components in the sample (electrokinetic bias). However, correcting each individual peak area with the internal standard rectifies this trend (Figure 5B) and proves the validity of the method in the presence of a fluctuating matrix composition. Sub-ppb Calibrations. Figure 6 shows the linearity plot of chloride (corrected area) obtained under t-ITP stacking conditions for a concentration range of 500-5000 ppt. A comparison with the corresponding data set of molybdate (Figure 7) indicates that Analytical Chemistry, Vol. 70, No. 11, June 1, 1998

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Table 2. Quantitative Analysis of Anions in the Presence of a Conductive Sample Matrix Composed of 1 ppm Ammonia and 50 ppb Hydrazine anion found (added), ppb

sulfate molybdate

sample 1

sample 2

sample 3

sample 4

sample 5

sample 6

4.09 (4.06) 5.81 (6.43)

3.58 (3.26)

2.21 (2.07) 5.81 (6.90)

0.33 (0) 1.70 (1.99)

5.03 (4.92) 8.01 (7.87)

8.13 (6.82) 9.94 (9.75)

Figure 6. Linearity plot of chloride (corrected areas). Each calibration point average of three consecutive runs (error bars, RSD of area counts; open circles: chloride-water blank subtracted from chloride peak area). Calibration standards/range, chloride, sulfate, molybdate (500-5000 ppt, each as indicated); sample preparation, internal standard (tungstate, 15 ppb), terminator (octanesulfonate, 2 ppm) added to each calibration standard; electrolyte, 50 mM boric acid, 20 mM LiOH, 0.1 mM TTAOH, 0.75% (w/w) Triton X-100, at pH 8.9; capillary, 50 µm × 60 cm; preinjection of 0.01 M LiOH, 50 mbar × 18s; injection, -5 kV × 24 s; separation at -14 µA.

Figure 5. Electrokinetic injection of 10 ppb of chloride under changing matrix concentration (ammonia 0-2000 ppb, hydrazine 0-100 ppb); (A) absolute areas; (B) peak area corrected with internal standard. Each point average of three consecutive runs (error bars, RSD of area counts). Sample, chloride, sulfate, molybdate, 10 ppb each; sample preparation, internal standard (tungstate, 30 ppb), terminator (octanesulfonate, 5 ppm) added to each sample; electrolyte, 50 mM CHES, 20 mM LiOH, 0.03% (w/w) Triton X-100, pH 9.2; capillary, 50 µm × 60 cm, preflushed with 1mM CTAB for 24 s at 2000 mbar; preinjection of 0.01 M LiOH, 50 mbar × 18 s; injection interval, -5 kV × 6 s; separation at - 25 kV.

the 500 and 1000 ppt data points for chloride are beyond the suggested linearity criterion. The linearity of chloride can be improved somewhat by subtracting the water blank profiles from each individual chloride profile (open circles in Figure 6). In the demonstrated example, we took the average of three water blank profiles (chloride location) and subtracted the area from the corresponding average of the obtained chloride sample profiles. This operation moves the 500 and 1000 ppt points closer to the 2266 Analytical Chemistry, Vol. 70, No. 11, June 1, 1998

Figure 7. Linearity plot of molybdate (corrected areas). Each calibration point average of three consecutive runs (error bars, RSD of area counts). Calibration standards (range), chloride, sulfate, molybdate (500-5000 ppt each as indicated); sample preperation, internal standard (tungstate, 15 ppb), terminator (octanesulfonate, 2 ppm) added to each calibration standard; electrolyte, 50 mM boric acid, 20 mM LiOH, 0.1 mM TTAOH, 0.75% (w/w) Triton X-100, at pH 8.9; capillary, 50 µm × 60 cm; preinjection of 0.01 M LiOH, 50 mbar x 18 s; injection, -5 kV × 24 s; separation at -14 µA.

(5% linearity criterion. For truly accurate measurements, at these low levels it is nonetheless necessary to eliminate all sources of external contamination (atmosphere, liquid handling, ionic trace levels in bulk chemicals, etc.) which can significantly bias the peak

Table 3. Quantitative Analysis of Anions between 0.5 and 5 ppb anion found (added), ppt

chloride nitrate sulfate molybdate fluoride phosphate

sample 1

sample 2

sample 3

sample 4

sample 5

sample 6

165 (0) 2920 (2000) 4792 (5000) 576 (500)

571 (500) 3036 (3000) 2788 (3000) 873 (1000)

1383 (1500)

1894 (2000) 1624 (1000) 1398 (1500) 1496 (1500)

3731 (3000)

5002 (5000)

1907 (2000) 3195 (3000) 1904 (2000)

2282 (2000)

3073 (3000)

5230 (5000)

596 (1000) 5076 (5000)

area count for ubiquitous ions as chloride and sulfate. Accurate ion analysis at or below ppb levels requires cleanroom facilities or, if available, on-line analysis instrumentation. Quantitative Analysis of Anions in the Range of 0.5-5 ppb (500-5000 ppt). In the following experiment, we applied the analysis protocol to recalculate known sample concentrations made up of chloride, nitrate, sulfate, molybdate, fluoride, and phosphate ranging from 0.5 to 5 ppb. The instrument was twopoint calibrated using a water blank (2 ppm octanesulfonate, 15 ppb tungstate in 18-MΩ water) and a 10 ppb standard of the above ions, with 2 ppm terminator and 15 ppb internal standard added to each solution correspondingly. No matrix ions were present in this experiment. Assuming linearity over the range as indicated previously, three data points were averaged and interconnected by the data analysis software in a point-to-point mode. After the calibration, all samples were analyzed in a single run without any averaging of peak areas. The amounts are calculated directly by the analysis software and displayed in Table 3. Good matches are obtained for molybdate being the “sanity check” of the analysis method. The determined chloride and sulfate amounts are acceptable, given the ubiquitous nature of these ions. The analysis of nitrate is due to the presence of NOx pollutants in the atmosphere, a problem in urban environments, which adds to the bias of the true peak profile. The precision of the values could be potentially improved by averaging several runs, which was not performed in this experiment. Reproducibility. A representative set of migration times and peak area reproducibilities of selected anions are listed in Table 4. The relative standard deviations (RSD, %) are computed on three consecutive runs. While the precision of the migration times is found to be ∼0.2% RSD and relatively independent of the sample concentration, the RSDs for the peak areas typically increase at lower concentrations. This observation can be explained by events of random contamination in the case of chloride. In the absence of any added sample, the residual chloride profile fluctuates at ∼14%, thus reflecting random contamination bias at this level.

1786 (5000)

1092 (1500)

Table 4. Reproducibility of Peak Areas and Migration Times for Chloride and Molybdate as a Function of Their Concentration RSD, % concn, ppt

chloride migration

chloride peak area

molybdate migration

molybdate peak area

0 500 1000 3000 5000

0.228 0.049 0.016 0.112 0.043

14.0 10.7 9.4 2.9 3.2

0.034 0.045 0.137 0.065

11.9 0.5 0.6 2.5

Molybdate as a nonubiquitous ion shows good precision for the peak areas of the 1 and 3 ppb data points. The comparably higher RSD for the 500 ppt level is probably due to random profile fluctuations for molybdate close to its detection limit. Conclusion. Capillary electrophoresis with conductivity detection combined with t-ITP stacking is demonstrated to be a sensitive and linear technique for quantitative trace ion analysis. The linear relationship between peak area and concentration has been evaluated by means of an ASTM linearity criterion on absolute and corrected peak areas. Consequent use of an internal standard and peak area correction increases the system linearity and precision of results and compensates for fluctuations in the matrix composition. The conductivity/CE system can be externally calibrated to conveniently determine unknown sample concentrations. Practical limitations of a benchtop CE system used for quantitative trace-level ion analysis rather arise from contamination bias from aerosol ingress and sample handling procedures, than being limited by the total system sensitivity. The experiments suggest that measurable contamination bias is introduced at levels equal or below 1 ppb sample ion. Received for review November 3, 1997. Accepted March 17, 1998. AC9712106

Analytical Chemistry, Vol. 70, No. 11, June 1, 1998

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