Anal. Chem. 1997, 69, 1169-1173
On-Line Dialysis Coupled to a Capillary Electrophoresis System for Determination of Small Anions Petr Kuban and Bo Karlberg*
Department of Analytical Chemistry, Stockholm University, S-106 91 Stockholm, Sweden
On-line dialysis performed in a flow injection analysis (FIA) system has been integrated with a capillary electrophoresis (CE) system via a specially designed interface. Samples are continuously pumped into a dialysis unit and the outgoing acceptor stream containing the analytes is allowed to fill a rotary injector in the FIA part of the system. A discrete, representative volume of the acceptor stream is injected into an electrolyte stream which continuously passes through the FIA-CE interface into which the end of a capillary has been inserted. An infinitesimal fraction of the injected acceptor plug is introduced electrokinetically when it passes the end of the capillary. Multiple sample injections are possible in one electrophoretic run, and the entire analytical procedure can easily be mechanized. The repeatability is in the range 1.6-3.3% (n ) 7). A wide range of real samples with complicated matrices (milk, juice, slurry, liquors from pulp and paper industry) was successfully analyzed without any off-line pretreatment using the fully mechanized system. Capillary electrophoresis (CE) is a versatile analytical technique used for the determination of all types of analytes ranging from small inorganic anions to large molecules such as proteins and fragments of nucleic acids and even viruses, cells, and particles.1 In spite of its versatility, off-line sample pretreatment steps such as extraction, filtration, centrifugation, or dilution are still required before the sample can be introduced into the CE system.2,3 These steps are characterized by being time consuming, having low precision, showing analyte losses, and involving manipulations including manual handling of toxic organic reagents and solvents. Increasing demands on the economy and safety of the total analytical procedure have led to the development of rational and fully mechanized cleanup and pretreatment techniques. However, these techniques are not always easily integrated with subsequent steps in the analytical chain. On-line dialysis is an elegant technique by which at least a few of these problems can be solved.4 In a commonly applied version, two phases, the donor and the acceptor phases, are separated by a semipermeable membrane. The analytes of interest are in most (1) Foret, F.; Krivankova, L.; Bocek, P. In Capillary Zone Electrophoresis; Radola, B. J., Ed.; VCH: Weinheim, Germany, 1993. (2) Schmitt, M.; Saulnier, F.; Malhautier, L.; Linden, G. J. Chromatogr. 1993, 640, 419-424. (3) Chiari, M.; Nessi, M.; Carrea, G.; Rioghetti, P. G. J. Chromatogr. 1993, 645, 197-200. (4) Van de Merbel, N. C.; Hageman, J. J.; Brinkman, U. A. T. J. Chromatogr. 1993, 634, 1-29. S0003-2700(96)00921-3 CCC: $14.00
© 1997 American Chemical Society
cases small anions and cations. These ions are transferred into the acceptor stream and will thus leave particles, colloids, and large molecules in the donor stream. On-line dialysis can be performed either in an air-segmented flow mode or in a flow injection analysis (FIA) arrangement. A fully selective transfer of only the analyte into the acceptor stream cannot be expected. Consequently, incorporation of at least one selective step is necessary. Selective reagents can be added to the acceptor stream, or selective detectors can be employed for this purpose. The FIA dialysis system can also be combined with an LC system to enhance selectivity. A fraction of the acceptor stream is then injected via a chromatographic valve into the LC system. This technique has been demonstrated for both inorganic5 and organic6 ionic species in blood serum, for other biological samples (muscle, liver extracts, tissues),7 and for food products (milk, eggs, meat).8 Bao and Dasgupta have shown that it is possible to combine dialysis with CE.9 They used a narrow-bore tubular dialysis membrane to join two capillaries. The distance between the capillary ends and the inner diameter of the tubular membrane defined the acceptor phase volume. The sample was then pumped on the outside of the membrane tube, and after a certain dialysis time, a high voltage was applied across the capillary. When all the constituents in the sample had reached the detector, dialysis of the next sample could be commenced. On-line coupling of in vivo microdialysis with CE has recently been described by Hogan et al.10 The microdialysis probe was implanted directly into the animal tissue. The dialyzate was pumped into a microinjection valve with a transferable volume of 60 nL. After injection, the sample plug was transported via a transfer capillary into a CE interface, where a fraction of the sample was electrokinetically introduced into the separation capillary. In the present paper, a new technique for coupling on-line dialysis in an FIA system to a CE system is described. The FIACE interface allows consecutive injections in one uninterrupted electrophoretic run.11 The developed technique has been successfully used for determination of anions in environmental (tap (5) Nordmeyer, F. R.; Hansen, L. D. Anal. Chem. 1982, 54, 2605-2607. (6) Turnell, D. C.; Cooper, J. D. H. J. Autom. Chem. 1985, 7, 177-180. (7) Andresen, A. T.; Rasmussen, K. E. J. Liq. Chromatogr. 1990, 13, 40514065. (8) Aerts, M. M. L.; Beek, W. M. J.; Brinkman, U. A. T. J. Chromatogr. 1988, 435, 97-112. (9) Bao, L.; Dasgupta, P. K. Anal. Chem. 1992, 64, 991-996. (10) Hogan, B. L.; Lunte, S. M.; Stobaugh, J. F.; Lunte, C. E. Anal. Chem. 1994, 66, 596-602. (11) Kuban, P.; Engstro ¨m, A.; Olsson, J. C.; Thorse´n, G.; Tryzell, R.; Karlberg, B. Anal. Chim. Acta 1997, 337, 117-124.
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water, soil extract, snow), food (milk, juice), and industrial (liquors from the pulp and paper industry) samples. The samples were introduced in the FIA part of the system without prior pretreatment. EXPERIMENTAL SECTION Chemicals, Solutions, and Samples. All chemical were of reagent grade. Deionized water was used throughout. Inorganic anion stock solutions, 10 000 ppm, were prepared from the corresponding sodium salts except for the fluoride stock solution, which was prepared from its potassium salt. Organic anion stock solutions were prepared from the acids, which were neutralized by using sodium hydroxide. All multi-ion calibration solutions were prepared from these stock solutions. The electrolyte buffer contained 6 mM sodium chromate, 3.2 × 10-5 M cetyltrimethylammonium bromide (CTAB), and 3 mM boric acid and it was prepared daily from individual stock solutions: 600, 50 (5% v/v acetonitrile), and 100 mM, respectively. The pH was adjusted to 8.0 by addition of a 100 mM sodium borate solution. All real samples were introduced into the FIA system without prior treatment; however, the liquor samples from the pulp and paper industry were diluted 1:100 or 1:1000 with water. Instrumental. FIA System. An eight-channel peristaltic pump (Gilson, Villiers le Bel, France) was used for the continuous delivery of electrolyte and for pumping acceptor and donor solutions. A one-channel injection valve with a 50 µL sample loop was controlled through a FIAstar 5020 unit (Perstorp Analytical, Ho¨gana¨s, Sweden). All parts of the FIA manifold system were connected via 0.7 mm i.d. PTFE tubings. Cuprophan dialysis membranes were used in a commercial dialysis unit (5106 dialysis module, Perstorp Analytical). FIA-CE Interface. The new FIA-CE interface earlier presented by Kuban et al.11 was used for sample introduction into the separation capillary of the CE part of the system. The anions were introduced electrokinetically. CE Part. The CE part of the system was accommodated in a homemade plexiglass box equipped with a safety lock on the access door for protection. It consisted of a high-voltage (HV) supply (Series 320, Bertan Associates Inc., Hicksville, NY) and two platinum electrodes positioned as shown in Figure 1B. A potential of 25 kV was applied during all runs. Polyamide-coated capillaries (50 µm i.d.; 375 µm o.d.) (Polymicro Technologies, Phoenix, AZ) were used. The total length of the capillary was 80 cm and the length from injection to detection was 45 cm. Detection Part. An Isco CV4 UV-visible detector (Isco, Lincoln, NE) was used for indirect UV detection at 372 nm. Electropherograms were registered using an ELDS 900 laboratory data system (Chromatography Data Systems, Kungsho¨g, Sweden) on an IBM personal computer. Principle. Figure 1B shows the complete flow scheme of the FIA-CE dialysis device. The sample, S, is pumped into the FIA part of the system and into the dialysis unit. An acceptor stream of water, A, is used. The analyte anions penetrate the membrane, M, and enter into the acceptor stream. The acceptor stream is led into the injector, and the injector loop is filled (V1 position). When the loop has been filled completely with representative sample, injection takes place and the sample is transferred to the electrolyte, E (V2 position). The injected sample then passes by the end of the capillary situated in the FIA-CE interface and a small fraction is electrokinetically introduced into the capillary 1170 Analytical Chemistry, Vol. 69, No. 6, March 15, 1997
Figure 1. (A) Cross-sectional view of the FIA-CE interface and (B) schematic diagram of the FIA-CE system used for on-line sample dialysis: (S) sample, (A) acceptor stream, (E) electrolyte, (M) dialysis membrane, (D) UV detector, (V1) injection valve in filling position, (V2) injection valve in inject position, (W) waste, (C) capillary, (Pt) platinum electrodes, and (HV) high-voltage supply.
where separation takes place. The detector, D, is situated 45 cm from the introduction end of the capillary. Optimization of Flow Conditions. The Codex 2.5 software (SumIT System, Solna, Sweden) was used to create an experimental design. Ratios of donor and acceptor flow rates in conand countercurrent arrangements were chosen as variables, and the sensitivity was chosen as the response. The flow rates ranged from 0.5 to 3 mL/min; the levels were governed by practical conditions such as pressure buildup at flow rates above 3 mL/ min and speed of analysis at flow rates below 0.5 mL/min. Response surface plots of sensitivity as a function of flow rates of acceptor and donor streams revealed that the acceptor flow rate should be kept as low as possible. On the other hand, the donor flow rate should be as high as possible. No significant difference between sensitivity obtained in con- and countercurrent arrangements, respectively, was observed. Concurrent design was finally chosen since this arrangement caused a low membrane overpressure. Selected flow rates for the donor and the acceptor phases were 3.0 and 0.5 mL/min, respectively. RESULTS AND DISCUSSION Basic System Characteristics. Analysis of a Standard Solution Containing 24 Anions. A complete electropherogram of the 24-anion model solution is presented in Figure 2. No peak of ascorbate was observed. Ascorbic acid (ascorbate) is probably oxidized in the electrolyte solution, containing CrO42-, to some nonabsorbing product. For
Figure 2. Electropherogram obtained for the 24-anion standard mixture. Two consecutive injections (A) without and (B) with dialysis. Time interval between injections, 5 min. FIA-CE conditions: HV 25 kV; chromate buffer as electrolyte; flow rate of electrolyte and sample 3.0 mL/min; flow rate of acceptor stream 0.5 mL/min. Peaks: (1) S2O32-, (2) Cl-, (3) SO42-, (4) NO3-, (5) oxalate, (6) HS-, (7) SO32-, (8) citrate, (9) maleinate, (10) fumarate, (11) F-, (12) tartrate/ succinate/formate/malate, (13) HPO42-, (14) HCO3-, (15) acetate, (16) OH-, (17) propionate, (18) lactate, (19) butyrate, and (20) benzoate.
Figure 3. Repeatability of the dialysis. Seven consecutive injections; time interval between injections 30 s. FIA-CE conditions as in Figure 2. Peaks: (1) Cl-, (2) SO42-, and (3) NO3-. Table 2. Repeatability (rsd, n ) 7) anion ClSO42NO3-
peak ht, %
peak area, %
3.3 3.2 1.6
3.2 4.8 3.7
Table 1. Relative Transport Efficiency Values Obtained with the FIA-CE Dialysis Systema ion
rel transport effic, %
ion
ClOHNO3Fformate acetate HCO3propionate benzoate lactate HPO42SO32-
54 53 50 36 28 25 23 20 19 16 11 10
S2O32butyrate SO42HSmaleinate fumarate succinate oxalate malate tartrate citrate ascorbate
a
rel transport effic, % 10 9 8 7 7 6 6 4 2 2 1
Sample flow rate, 3 mL/min; acceptor flow rate, 0.5 mL/min.
anions, as well as large organic anions, displayed significantly lower values. Again, no ascorbate peak was observed. According to the Fick’s law
J ) -D(A/τ)(dc/dx)
the diffusion rate J (mol/s) of the analyte depends on its diffusion coefficient D (m2/s), on the concentration gradient -dc/dx (mol/ m4), and on the effective membrane area A (m2). The factor τ, the tortuosity of the membrane, is a constant that takes all other membrane parameters such as porosity, pore size, membrane thickness, etc., into account. The diffusion coefficient D can be expressed by the Stokes-Einstein relationship
D ) kT/6πηr ascorbic acid determination, preferably phosphate buffer and direct UV detection might be used as recommended by Chiari et al.3 Some of the peaks, especially the peaks of the organic anions maleinate, fumarate, malate, and succinate, are not fully resolved. These anions have similar structures. Interestingly, the peak of the OH- anion, which was supposed to have the fastest migration of all the selected anions, unexpectedly migrated very late. Such behavior was not observed in the chromate-based electrolytes in the absence of boric acid. Most probably the hydroxide ion interacts with the boric acid present in our electrolyte to form B(OH)4- anions which migrate even after the acetate ions. Transport Efficiency Studies. To evaluate the relative transport efficiency values of the selected set of 24 anions, a model mixture of 100 ppm of each anion was first dialyzed. The peak signals were then compared with signal values obtained for the same standard solutions (100 ppm) which were introduced without being subjected to dialysis in the FIA-CE system. The observed relative transport efficiency values for the 24 selected anions are summarized in Table 1. The values are listed in descending order. As expected, small inorganic anions, such as Cl- and OH-, had the highest transport efficiency values, while sulfur-containing
(1)
(2)
where k is the Boltzmann constant (J/K), T the absolute temperature (K), η the solvent viscosity (kg m-1 s-1), and r the radius of the analyte molecule. The diffusion rate at constant T and η depends only on the analyte diameter. Consequently, the estimation of transport efficiency (or diffusion rate) for various analytes can be based on respective ionic or molecular size values. Small inorganic anions are expected to have several times higher transport efficiency than large organic anions. Repeatability. Repeatability of the FIA-CE dialysis system was evaluated by using a standard solution containing 100 ppm each of the anions Cl-, SO42-, and NO3-. This standard solution was introduced in the system as solution S as shown in Figure 1B. The dialyzate was repeatedly injected into the FIA-CE system every 30 s. The electropherograms obtained are shown in Figure 3. The calculated repeatability values are given in Table 2. The repeatability is expressed as the rsd values of the peak heights and the peak areas obtained for seven injections. Real Samples. Tap Water. Tap water from two different sources was analyzed both with and without dialysis incorporated in the flow system. Electropherograms of three consecutive Analytical Chemistry, Vol. 69, No. 6, March 15, 1997
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Figure 5. Analysis of a mud sample. Time interval between the two injections 3.5 min. FIA-CE conditions as in Figure 2. Peaks: (1) Cl-, (2) NO3-, and (3) HCO3-.
Figure 4. Analysis of tap water. Three injections were made without dialysis followed by three injections with dialysis. Time interval between injections 25 s. (A) tap water I; (B) tap water II. FIA-CE conditions as in Figure 2. Peaks: (1) Cl-, (2) SO42-, (3) NO3-, and (4) HCO3-.
injections made are shown in Figure 4. The detectable anions were Cl-, SO42-, NO3-, and HCO3-. Some interesting observations were made. First, multiplicate injections of the same sample can be carried out before all the constituents in the previously injected portions of the sample had reached the detector. Anion peaks can mingle still allowing quantitative evaluation of all peaks. No shifts of migration times or peak deterioration were registered. However, this approach should be used with care and only applied for samples with “known” compositions for which the risk of overlapping peaks has been shown to be insignificant. Second, even if the amount of NO3- anion was very small in the tap water sample, dialysis did not significantly lower the limit of detection. Analysis of the tap water requires in general no sample pretreatment. However, the dialysis step becomes necessary when other types of water are analyzed, such as process water containing particles that may clog the conduits in the FIA system. An in-line filter can then be incorporated into the inlet to the dialysis unit. Road Snow and Mud Samples. Road snow and mud samples containing dust particles and gravel were collected. The snow samples were melted and introduced twice in the FIA-CE dialysis systems. Mud samples were dispensed in water and mixed before introduction into the system. A typical electropherogram is depicted in Figure 5. The peaks are easily identified by using standards. No membrane fouling was observed. Milk and Juice. Electropherograms for dialyzates of orange juice and milk are shown in panels A and B of Figure 6, respectively. The juice sample contained, in addition to the 1172 Analytical Chemistry, Vol. 69, No. 6, March 15, 1997
Figure 6. Analysis of orange juice containing fruit pulp (A) and milk (B). Time interval between two injections of dialyzed sample 3.5 min. FIA-CE conditions as in Figure 2. (A) Peaks: (1) Cl-, (2) SO42-, (3) citrate, (4) malate/succinate, (5) HCO3-, and (6) benzoate. (B) Peaks: (1) Cl-, (2) SO42-, (3) citrate, (4) HPO42-, (5) HCO3-, (6) lactate, and (7) butyrate.
common inorganic anions Cl-, SO42-, and HCO3-, large amounts of citrate and some malate and succinate. The peaks of the last two anions are not separated, probably due to the similarity in structure as discussed before. A small peak of benzoate also appeared. Note that the second injection can be made before all peaks from the first injection have eluted. This possibility enhances the sampling throughput considerably. Comparison of these results with results obtained for manually treated samples could not easily be made. The juice sample was a commercially available product and it contained large amounts of fruit pulp, which did not allow direct injection not even after filtration since the sample was still opaque. Similar problems also occurred for the milk sample.
Liquors from the Pulp and Paper Industry. Samples of black, green, and white liquors were obtained from the Swedish Pulp and Paper Research Institute, Stockholm, Sweden. The samples were diluted 100 times and introduced into the FIA-CE dialysis system. As expected, the white and green liquor samples contained inorganic anions only, while some organic anions were present in the black liquor. The OH- anion appeared after acetate in the electropherograms as observed previously; the higher the pH, the larger the OH- peak. Nine anions could readily be identified in black liquor, and further studies will be conducted to evaluate this method for process control purposes. CONCLUSIONS On-line dialysis in an FIA arrangement is a favorable technique for the pretreatment of samples with a complicated matrix composition before introduction on a CE system. The direct coupling of on-line dialysis to a CE system has been demonstrated. The resulting FIA-CE system represents a powerful analytical tool, which can be used to analyze a huge variety of aqueous samples available in relatively large volumes. Multiple analyte determination is made possible in one, uninterrupted electrophoretic run. Environmental, food, and industrial samples con-
taining dust particles, clay, little stones, fruit meat, proteins, and macromolecules have successfully been analyzed without any need of off-line pretreatment. The developed technique might have a large potential for process analysis. The high speed of the analysis, comparable to that of the FIA technique, is a prerequisite for continuous on-line monitoring. The specially designed interface enables consecutive sampling without any need to wait until all the constituents in the previously injected sample reach the detector. ACKNOWLEDGMENT The authors thank Fernando Alvorado from the Swedish Pulp and Paper Research Institute, Stockholm, for providing the liquor samples from the pulping process and the Swedish Institute for financial support for P.K.
Received for review September 11, 1996. December 20, 1996.X
Accepted
AC960921L X
Abstract published in Advance ACS Abstracts, February 1, 1997.
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