Anal. Chem. 1994,66, 1817-1824
On-Line Isotachophoretic Sample Pretreatment in Ultratrace Determination of Paraquat and Diquat in Water by Capillary Zone Electrophoresis D. Kaniansky,'lt F. Ivhyi,t and F. I. Onuska* Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University, 842 15 Bratislava, Slovakia, and Research and Application Branch, National Water Research Institute,* 867 Lakeshore Road, P.O. Box 5050, Burlington, Ontario L7R 4A6, Canada
Capillary zone electrophoresis (CZE) with on-line isotachophoretic (ITP)sample pretreatment in a column-coupling configuration of capillary electrophoresisequipmentwas studied from the point of view of (u1tra)traceanalysis of paraquat and diquat in water. Using this technique, a sample volume as large as 90 wL can be accommodated and the herbicides can be analyzedwith a detectionlimit of mol/L. A photometric absorbance detector was used operating at 310-nm wavelength. Adsorption losses of the pesticides on the walls of the sample containers were found to be main sources of analytical errors. They could be eliminated by spiking the samples with diethylenetriamine. Capillary electrophoresis (CE) techniques have been shown to be promising alternatives to the separation and/or analysis of various ionogenic pesticides or their transformation products. The use of capillary zone electrophoresis (CZE),1.2micellar electrokinetic chromatography (MEKC),3.4 and capillary isotachophoresis (ITP)S-12appears to be particularly promising. In general, these techniques provide very low limits of detection (e.g., femtomole levels for on-column UV photometric detectors). Their use for the analysis of pesticides in environmental matrices is hindered by interfering ionic matrix constituents. The use of suitable sample pretreatment techniques is, therefore, essential for trace analysis applications of CE. Liquid-liquid extraction8J0 and solid-phase extraction6,13have been shown to offer simple solutions for removal of interferences. A solid-phase extraction technique would appear to be very convenient for an on-line coupling to CE separation systems13J4 and for the automation of sample preparation. ~~~
~
t Comenius University. t National Water Research Institute. 1 NWRI Contribution No. 93-69.
( I ) Foret, F.; Sustltek, V.; BoEek, P. Electrophoresis 1990, 11, 95-97. (2) Cai, J.; El Rassi, Z. J . Liq. Chromatogr. 1992, 15, 1193-1200. (3) Wu, Q.; Claessens, H. A,; Cramers, C. A.; Chromatographia 1992.34.25-30. (4) Cai, J.; El Rassi, Z. J. Chromatogr. 1992, 608, 31-45. ( 5 ) Kaniansky, D.; Madajovl, V.; Hutta, M.; Zilkovl, I. J . Chromatogr. 1984, 286, 395-406. (6) Hutta, M.; MarBk, J.; Kaniansky, D. J. Chromafogr. 1990, 509, 271-282. (7) Kenndler, E.; Kaniansky, D. J . Chromatogr. 1981, 209, 306309. (8) Strlnskf, Z. J . Chromarogr. 1985, 320, 219-231. (9) StrBnskf, Z.; Opllkovl, I. Acta Uniu. Palacki. Olomuc., Far. Rerum Nut. Chem. XXVI 1987.88, 149-157. (10) Dombek, V.; Ruiitka, E.; Strlnskf, Z. Acta Univ. Palacki. Olomuc., Fac. Rerum Nut. Chem. XXVII 1988, 91, 153-163. (11) Dombek, V.; StrBnskf, Z. J . Chromatogr. 1989, 470, 235-240. (12) Dombek, V. J . Chromatogr. 1991, 545, 427-435. (13) Cai, J.; El Rassi, Z. J . Liq. Chromatogr. 1992, 15, 1179-1192. (14) Prusfk, Z.; KaHiEka, V. J. Chromatogr. 1987, 390, 39-50. 0003-2700/94/0366-1817$04.50/0 0 1994 American Chemical Society
Sample pretreatment techniques based on extraction principles can usually concentrate analytes from large sample volumes with high recoveries. However, theenrichment factors for trace analytes15 may be less favorable when matrix constituents of similar physicochemical properties are present in the samples. Considering the amounts of the analytes currently required for CZEanalysis,16it is apparent that highperformance column separation techniques will accommodate adequate sample load and will serve as convenient sample pretreatment alternatives in such situations. The concentrating capability of CZE i t ~ e l f l or ~ -CZE ~ ~ in combination with in-column ITP focusing2G25can be adequate when preconcentration of the analytes during the pretreatment does not occur. High-performance liquid chromatography (HPLC)6 and preparative ITP5 have already proved very beneficial in this respect for the ITP analysis of residues of pesticides in soil extracts. For obvious reasons (a minimum number of sample handling steps, short analysis time) it is desirable that highefficiency sample preparation techniques be on-line coupled with final C E separations. Some instrumentation alternatives based on liquid chromatography sample preparation are already described in the l i t e r a t ~ r e . ~Column-coupling ~,~~ devices as designed for ITP28-30offer a promising solution for various C E techniques as demonstrated recently for the ITPCZE c ~ m b i n a t i o n . ~ l -A~ ~significant advantage of this approach in trace CZE analysis is that the ITP stage provides (15 ) Mizuike, A. Enrichment Techniques for Inorganic Trace Analysis: Springer-Verlag: Berlin, Heidelberg, New York, 1983; p 6. (16) Ewing, A. G.;Wallingford, R. A.; Olefirowicz, T. M. Anal. Chem. 1989,61, 292A-300A. (17) Mikkers, F. E. P.;Everaerts, F. M.; Verheggen, Th. P. E. M. J . Chromatogr. 1979, 169, 11-20. (18) Chien, R.-L.; Burgi, D. S.Anal. Chem. 1992, 64, 489A496A. (19) Beckers, J. L.; Ackermans, M. T. J. Chromatogr. 1993, 629, 371-378. (20) Hjerth, S.;Elenbring, K.; Killr, F.; Liao, J.-L.; Chen, A. J. C.; Siebert, C. J.; Zhu, M.-D. J . Chromatogr. 1987, 403, 47-61. (21) Verheggen, Th. P. E. M.; Schoots, A. C.; Everaerts, F. M. J . Chromatogr. 1990,503, 245-255. (22) Dolnfk, V.; Cobb, K. A.; Novotny, M. J . Microcolumn Sep. 1990,2,127-131. (23) Foret, F.; SustlEck, V.; BoEek, P. J . Microcolumn Sep. 1990, 2, 229-233. (24) Schwer, C.; Lottspeich, F. J. Chromatogr. 1992, 623, 345-355. (25) Thompson, T. J.; Foret, F.; Vouros, P.; Karger, B. L. Anal. Chem. 1993.65, 9OC-906. (26) Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1990, 62, 978-984. (27) Yamamoto, H.; Manabe, T.; Okuyama, T. J . Chromatogr. 1989,480, 277283. (28) Everaerts, F. M.; Verheggen, Th. P. E. M.; Mikkers, F. E. P. J. Chromatogr. 1979,169, 21-38. (29) Kaniansky, D. Thesis, Comenius University, Bratislava, 1981. (30) Dolnfk, V. Thesis, Institute of Analytical Chemistry, Czechoslovak Academy of Sciences, Brno, 1987. (31) Kaniansky, D.; Marlk, J. J . Chromatogr. 1990, 498, 191-204.
Analytical Chemistry, Vol. 66,No. 11, June 1, 1994
1817
a high enrichment factor for the analyte(s) by combining the inherent concentrating power of the ITP separation39with a well-defined removal of the matrix constituents from the separation compartment.38 Promising results obtained in the quoted works led us to investigate the potential of the ITP-CZE tandem in the analysis of ionogenic pesticides which need to be monitored at trace concentration levels in water. Paraquat and diquat, widely applied general herbicides for terrestrial and aquatic plants, were selected for this investigation. At present their analyses in water are carried out by gas chromatography40 or by HPLC.41,42Recently, the US.Environmental Protection Agency introduced a method based on HPLC combined with solid-phase e ~ t r a c t i o n .This ~ ~ method has detection limits of 0.44 pglkg for diquat and 0.80 pglkg for paraquat using a 250-mL sample volume.42 For known reasons,w6 CZE, although providing resolution of these herbicides in the femtomole range,2has limited capabilities in their direct trace analysis in samples containing ionic matrix constituents at 10-3-1 0-2 mol/L concentrations. Similar conclusions can be drawn for ITP with conductivity detection.8J0 In this work we show that the ITP-CZE tandem in the column-coupling configuration of the separation unit enables the determination of paraquat and diquat at 10-9-10-8 mol/L concentrations in tap and surface waters when the electrolyte system provides their separation from matrix macroconstituents in the ITP stage. It is also shown that adsorption losses of the analytes in the sample storage containers may be responsiblefor serious analytical errors at theseconcentrations when appropriate measures are not taken.
EXPERIMENTAL SECTION Instrumentation. An instrument for column-coupling C E with a hydrodynamically closed separation system as schematically shown in Figure 1 was assembled from the following units and components: (i) A DA-30 high-voltage power supply (Spectrovision, Chelmsford, MA) was switched into the current stabilized mode. The driving currents were 250 and 200 pA in the ITP and CZE stages, respectively. (ii) A sample injection system for ITP suitable for valve (a 25-pL internal sample loop) and microsyringe injections or for their combinations was obtained from Labeco-Villa (SpiSskB Nova Ves, Slovakia). (32) Stegehuis, D. S.; Irth, H.; Tjaden, U. R.; van der Greef, J. J . Chromatogr. 1991, 538, 393-402. (33) Kiivlnkovl, L.; Foret, F.; BoEek, P. J. Chromatogr. 1991, 545, 307-313. (34) Stegehuis, D. S.; Tjaden, U. R.; van der Greef, J. J. Chromarogr. 1992,591, 341-349. (35) Foret, F.; Szoko, E.; Karger, B. L. J. Chromatogr. 1992, 608, 3-12. (36) Reinhoud,N. J.;Tinke,A.P.;Tjaden,U.R.;Niesen, W. M.A.;vanderGreef, J. J . Chromarogr. 1992, 627, 263-271. (37) Hirokawa, T.; Ohmori, A.; Kiso, Y . J . Chromatogr. 1993, 634, 101-106. (38) Kaniansky, D.; Marlk, J.; Madajovi, V.; Simunibvi, E. J. Chromatogr. 1993, 638, 137-146. (39) Everaerts, F. M.;Bcckers, J. L.; Verheggen, Th. P. E. M.; Isotachophoresis; Elsevier: Amsterdam, 1976. (40) Cannard, A. J.; Criddle, W.J. Analyst 1975, 100, 848-853. (41) Simon, V. A,; LC-GC 1987, 5, 899-903. (42) Simon, V. A.; Taylor, A. J . Chromatogr. 1989, 479, 153-158. (43) United States Environmental Protection Agency. Method 549, Determination of Diquat and Paraquat in Drinking Water by Liquid-Solid Extraction and HPLC with Ultraviolet Detection; July 1990. (44) Beckers, J. L.; Everaerts, F. M. J. Chromatogr. 1990, 508, 3-17. (45) Beckers, J. L.; Everaerts, F. M. J. Chromatogr. 1990, 508, 19-26. (46) Gebauer, P.; Thormann, W.; BoEck, P. J. Chromatogr. 1992, 608, 47-57.
1818
Analytical Chemistry, Vol. 66,No. 1 1, June I , 1994
HV
Flaurr,1. Schematicof the separationunit for capillary electrophoresis with oaupled columns: 1 = terminatfng electrolyte compartment; 2 = sample injection system for valve (SJand microsyringe(S,) sample Introduction; 3 = septum; 4 = ITP column with FEP capillary tube (5) and on-column conductivii cell (6); 7 = bifurcation block for coupllng the columns; 8 = refllllng blocks; 9 = CZE column with fused slllca capillary tube (10); 11 = photometric detectlon cell; 12 = needle valves for tight closing of the separationcompartment;13 = cellophane membrandir; 141Tp,14= = counter-electrode compartments for the ITP and C2E columns, respectively; 15 = driving electrodes: HVR = hlgh-vobge reley (connected to the ground pole of the high-voltage power supply (HV)); le, ce = connections for the refillingof the columns with the leading and carrier electrolytes, respectively.
(iii) An ITP column provided with a 0.85-mm-i.d. (80 mm in the length) capillary tube made of fluorinated ethylenepropylene (FEP) copolymer with an on-column conductivity sensor was from Labeco-Villa. (iv) Counter-electrode compartments for the ITP and CZE columns with the refilling blocks were from Labeco-Villa. (v) A bare fused silica capillary tube (0.25” i.d.; 0.37mm 0.d.) used as a CZE column was obtained from Polymicro Technologies (Tucson, AZ). The length of the tube was 350 mm, and its length from the start to the detector was 250 mm. (vi) A laboratory-made conductivity detector similar in principle to that described in the literature (ref 39, p 148) was used to monitor the separation in the ITP stage. (vii) An AD-2000 absorbance detector (Spectrovision) was used to monitor the CZE separations with a 0.24 response time. The capillary tube was inserted into the detection cell as recommended by the detector manufacturer. (viii) A high-voltage relay (Labeco-Villa) was used for a manual switching of the columns with a precision of 0.3 s (when used under computer control, the precision is 0.01 s). (ix) A line recorder (Philips, Eindhoven, The Netherlands) was used to register the signals from the detectors. Safety Considerations. This laboratory-assembled CE equipment involves theuseof high potentials. The high-voltage unit was provided with a laboratory-constructed adjustable trip switch disconnecting the delivery of the driving current at a preselected potential. Extreme caution in the handling of the equipment is, however, needed. Chemicalsand Stock Solutions. Morpholinoethanesulfonic acid (MES) and N-(2-acetamido)-2-aminoethanesulfonicacid
(ACES) were from Sigma (St. Louis, MO). Tris(hydroxymethy1)aminomethane (Tris), potassium hydroxide, diethylenetriamine (DETA), triethylamine, citric acid, and tetrahexylammonium bromide were from Aldrich (Milwaukee, WI). 1,l’-DimethyL4,4’-bipyridiniumdichloride (paraquat) and l,l’-ethylene-2,2’-bipyridiniumdibromide (diquat) were obtained from ChemService (West Chester, PA). Demineralized aqueous solutions (0.5% w/v) of methylhydroxyethylcellulose 30 000 (MHEC) and poly(ethy1ene glycol) 5 000 000 (PEG) obtained from the Laboratory of Environmental Analysis (Faculty of Science, Comenius University, Bratislava, Slovakia) added to the electrolyte solutions served as anticonvective additives. The remaining chemicals were purchased from Fisher (Raleigh, NC). The chemicals were used for preparations of the solutions as received. Demineralized water from a Milli-Q System (Millipore, Bedford, MA) was used for the preparation of the solutions. A stock solution of DETA (79.8 X mol/L) was prepared by dissolving the preparative (99% purity) in water. A stock solution of the herbicides contained paraquat at mol/L and diquat at 4.23 X mol/L 5.63 X concentrations. The diquat was taken to be the monohydrate and the paraquat to be the tetrahydrateS4* Cleaning of Sample Containers. 1. Procedure A. The samplecontainers were washed three times with a 0.05 mol/L aqueous solution of citric acid. Then they were repeatedly washed and filled with Milli-Q purity water until they were ready to be used. The containers were washed three times with a sample to recondition the surface before storage. 2. Procedure B was the same as procedure A except that in the last wash in the reconditioning step 20 pL of the stock solution of DETA was added. Samples. Tap water samples were collected from the local water supply into 100-mL sample containers made of Nalgene (cleaned as described above). The required amounts of the spiking herbicides were added from their stock solution by a 701N Hamilton microsyringe (Hamilton, Reno, NE) directly or via one dilution step. When required, an appropriatevolume (1-20 pL) of the stockDETA solution was added to the sample. Lake Ontario water samples were also collected into cleaned containers made of Nalgene. They were spiked with appropriate amounts of the herbicides in the same way as for tap water. A 3-pL volume of the stock DETA solution per 100 mL of the sample was added. The lake water samples were filtered through a 0.45-pm syringe filter (Gelman, Ann Arbor, MI) before the injection into the instrument.
RESULTS AND DISCUSSION Separation and ITP Sample CleanupConditions. CZE with on-line ITP sample pretreatment performed in the columncoupling configuration of the separation unit can be used in several working mode^.^',^^ Each of these modes consists of a well-defined sequence of separating, concentrating, and sample cleanup steps. The choice of the working mode is closely related to the type of the analytical problem to be solved, and the one preferred in this work consists of the following steps: (i) ITP separation of paraquat and diquat from the cationic sample macroconstituents (Mg2+,Ca2+,Na+, and K+).The
Tris /
/
20 s P,D,DETA Na,Mg,Ca
.
Tris /
/
lR
t Flgure 2. Isotachopherograms from the separation of cations in tap water. The records were obtained in the electrolyte systems ITP-2 (a) and ITP-1 (b). In both instancesthe sample volumes were 85 pL. R, t= increasingresistanceand time, respectively. Thedrlvingcurrent was 250 pA.
separation is accompanied by a defined concentration of the herbicides into a narrow zone. Simultaneously, the sample constituents having effective mobilities higher than or equal to that of the leading ion are removed from the separation compartment. (ii) Removal of the sample constituents migrating between the leading zoneand the zone of herbicides from the separation compartment after the ITP stage. (iii) Transfer of the sample fraction of analytical interest (containing, besides the herbicides, only a restricted and, in terms of migration characteristics, well-defined group of the sample constituents4’) into the C Z E stage. (iv) CZE separation and detection of the transferred sample constituents. The choice of the constituents of the electrolyte systems followed the above scheme, and it was made with a strong emphasis on a high sample load. To achieve this, the composition of the leading electrolyte should maximize relative differences in the effective mobilities of the analytes and the matrix macroconstituents to work with a high resolution rate48 in the ITP stage. From previous works dealing with ITP separations of paraquat and diquat7.8.10 it can be deduced that the leading electrolyte containing counterionic constituents of higher charge numbers49 offers a favorable solution. A comparison of citrate and MES, under otherwise identical working conditions, undoubtedly confirms these expectations (Figure 2). Here, the former counterionic constituent retards the herbicides relative to Na+ ions and considerably reduces the effective mobilities of Ca2+ and Mg2+ ions via complex formation.50 Its use in the leading electrolyte (system ITP-1, Table 1) enabled us to inject sample volumes as large as 90 (47) Marhk, J.; LaHtinec, J.; Kaniansky, D.; Madajovh, V. J . Chromorogr. 1990, 509, 287-299. (48) Mikkers, F. E. P.; Everaerts, F. M.; Peck, J. A. F. J . Chromorogr. 1979,168, 293-3 15. (49) Kaniansky, D.; Madajovh, V.; Zelenskq, I.; Stankoviansky, S. J . Chromatogr. 1980, 194, 11-19. (50) Hirokawa, T.; Kiso, Y. J . Chromafog. 1982, 248, 341-362.
Analytical Chemistty, Vol. 66, No. 11, June 1, 1 9 9 4
1819
Table 1. Electrolyte Systems
ITP-1
parameter solvent cation concn (mMj counterion PH additive concn ( p r , wiv)
solvent cation concn (mM) counterion PH additive concn ( ' c , wi v)
1ead i n g
ITP-2 1ead in g
ITP-1, ITP-2 terminating
Hz0 K+
Hz0
10 citric 6.08 MHEC 0.2 ZE-!a carrier
10 MES 6.12 MHEC 0.2 ZE-lb carrier
H20 Tris+ 5 citric 7.80
Hz0
HzO
Hz0
Tris+ 10 citric 6.08 PEG 0.15
Tris+ 9.8 citric 6.08 PEG 0.15
Tris+ 10 MES (ACES) 6.10 PEG 0.15
K+
I
1
DETA
ZE-2 carrier
matrix macroconstituents (see also below) while for MES (system 1TP-2. Table 1 ) this volume was approximately 10 times lower. Paraquat and diquat (permanent double-charged cations) have very close ionic and a t the same time p H independent effective m o b i l i t i e ~ . * ~Therefore, ~ . ~ ~ ' ~ when weak organic bases are to be taken into account as sample constituents interfering in their analysis, the pH of the leading electrolyte provides an additional means for optimization of the separation and cleanup conditions. In this context, the p H value of the leading electrolytes employed in this work (Table 1 ) can be accepted as a compromise between the maximum attainable sample cleanup ( a high pH) and an efficient use of the delivered charge (minimized charge transport by very mobile H+ and O H - ions ) . From a description of the cleanup efficiency in twodimensional ITP.47it is apparent that the sample constituents should be transferred after the ITP pretreatment to a final separation in the ZE stage in a narrow mobility fraction. In two-dimensional ITP, this transfer is conveniently defined, e.g., via a pair of zones of sample macroconstituents or (more often) via a pair of zones of spacing constituents added to the sample. ,4n analogous approach appears suitable also for the ITP-CZE tandem.31,38In our case the herbicides preconcentrated with some of the matrix constituents into the boundary layer between the Na+ and terminating zones were transferred into the C Z E column for a final separation and detection (see also Figure 3). A rapid resolution of paraquat and diquat in the C Z E stage served as a criterion for our choice of the carrier electrolyte. In analogy with the ITP stage, we preferred the use of multiply charged counterionic constituents. Citrate chosen for this purpose provided a high resolution rate for the herbicides in the CZE stage (see Figure 3). When this constituent (migrating at pH = 6 in doubly and triply negativelj charged ionic forms) was replaced by MES or ACES (distributed at the same pH into uncharged and singly negatively charged forms). no resolution of the herbicides was obtained. Considering the C Z E separations of anions Analytical Chemistry, Vol. 66,
Tris
3
d
gL with a complete ITP resolution of the herbicides from the
1820
0 CZE
electrolyte
No. 11, June 1, 1994
Na\
\
r-
j
N 0
9
0
__*
t
225
265
16) Figure 3. Separation of paraquat (P) and diquat (D) in tap water. The sample (25-pL volume by valve injection) was spiked with paraquat and diquat at 10.6 X and 8.0 X mollL concentrations, respectively. The separationwas carried out in the electrolyte systems ITP-1 and ZE-lb. The photometric detector was set at a 310-nm wavelength. A dashed line with an arrow on the Na+ zone marks the fraction transferred into the CZE stage. The driving currents were 250 and 200 pA in the ITP and CZE stages, respectively. The time for the ITP preseparation was 6 min. Table 2. Separation Performance Characteristics for Paraquat and Diquat In the ZE Stagea constituent
parameterb migration time (sj S,($)
column efficiency (Nim) SI ( c c )
peak height (AU) s, ( p c ) peak area ( A U d Sr
(c,)
paraquat 248.6 0.76 545 000 30 46.7 X 14.6 1.6 X 6.9
diquat 258.4 0.50 195 000 20 32.2 X lo4 6.5 1.8 X 10-2 5.2
Tap water samples (25-pL volumes) spiked with paraquat (10.6 x 104mol/L) and diquat (8.0 X 1O4moliL) were repeatedlyanalyzed in the electrolyte systems ITP-1 and ZE-lb (Table 1). bFor all parameters, mean values from nine analysesare given. Nlm = number of theoretical plates per meter; s, = relative standard deviation. The column efficiency was calculated from the peak width a t half-height.
based on their differences in the charge numbers,51it seems reasonable to ascribe the resolution of the herbicides to their differences in ion-pair formation with citrate. Separation performance characteristics for the C Z E stage summarized in Table 2 show an unexpected difference in the separation efficiencies of the herbicides. Although no detailed investigation to explain this difference was carried out, we assume, from a comparison of analogous situations in CZE2a-24 and ITP-CZE c o m b i n a t i o n ~ ~ and I J ~ also . ~ ~ from descriptions of the ITP sample preconcentrations in a single-column CZE.44-46that Na+ ions transferred with the analytes into the C Z E stage (see Figure 3) exhibit a stronger focusing effect in this stage for paraquat than for less mobile diquat. Larger fluctuations in the peak heights of paraquat relative to those (51) Marlk, J.; Kaniansky, D.; SimuniEovl, E.; Madajovl, V . Book ofAbsrracrs. 8th International Symposium on Capillary Electrophoresis and IsotachophoreS I S . Rome, October 6-9. 1992; p 82.
* **
P /
P
/
P
,
/
P
I
b-
4
I
U
P
,P
P
/
,
/
/
/
L
P
x
,’
200
320
200
320
200
320
200
320
Figure 4. Adsorption of paraquat and diquat and the influence of DETA. A pair of tap water samples was spiked with paraquat (10.6 X moi/L) and diquat (8.0 X mol/L). Sample b contained in addition DETA at a moi/L concentration. The samples prepared simuitaneousiy were analyzed immediately after the preparation (2), with a 1-h delay (3), and after addition of DETA at a mol/L concentration (4). Note the recovery of the analytes on the addition of DETA to the sample (4, for sample a). The Separations were carried out in the electrolyte systems ITP-1 and ZE-la. The driving currents were 250 and 200 pA in the ITP and CZE stages, respectively. The photometric detection was performed at a 254-nm Wavelength. The time for the ITP preseparation was 6 min.
observed for diquat in the same experiments (Table 2), originating in variation in the amount of transferred Na+ ions which was caused by imprecision in the switching of the columns (see Experimental Section), also support this explanation. Sample Handling. It has been shown that paraquat and diquat can be lost as a result of adsorption on some surfacesw2 Therefore, the use of glass containers for storage of samples was excluded and containers made of Nalgene were employed in this work. While effective for ppm concentrations of the herbicides, this precaution was not sufficient to prevent serious analytical errors associated with adsorption losses at low ppbppt concentrations (see Figure 4). Various cationic constituents expected to preferentially accumulate on the surfaces were tested as sample additives to minimize these losses via a competitivebinding on the adsorption sites. Inorganic cations (Ca2+,MgZ+)at increased concentrations, organic bases known to suppress adsorption of basic compounds in liquid chrom a t o g r a p h ~ ,and ~ ~ compounds ,~~ changing electroosmoticflow in CZES4 were tested. Of these, DETA was found to be the most effective and electropherograms in Figure 4 show both the extent of adsorption losses and a positive role of DETA in their suppression. The addition of DETA to the samples not only was effective in preventing the adsorption of paraquat and diquat but was found to be responsible for contamination of the samples by displacing cationic constituents from the surfaces of inappropriately cleaned sample containers (Figure 5). While (52) Nawrocki, J. Chromafographia 1991, 31, 193-205. (53) Vervoort, R. J. M.; Maris, F. A.; Hindriks, H. J . Chromafogr. 1992, 623, 207-220. (54) Application Note, AN 68; Dionex Corp., Sunnyvale, CA, 1991.
P
@ L_
55
, D ,
,
hA’ 295
t(s) Figure 5. Sample contamination by using inappropriately cleaned sample container: (a) tap water containing DETA at a mol/L concentration (sample container cleaned by procedure B in the ExperimentalSection); (b)tap water as in part a but spiked with paraquat (10.6 X 104mol/L)anddiquat(8.0 X 10-8moi/L)inasamplecontainer cleaned as in part a; (c) tap water sample mixed from 51.O g of sample a and 36.0 g of sample b in a container cleaned by procedure A (X = impurities appearing in the mixed sample); (d) the same sample as in part c, but the detectionwavelength was 3 10 nm. Electropherograms a-c were registered at 254 nm. The separations were carried out in the electrolyte systems ITP-1 and ZE-la with driving currents 250 and 200 pA in the ITP and CZE stages, respectively. The time for the ITP preseparation was 6 min.
undetected when DETA was not present in the sample, the contaminants appeared immediately on its addition at a 1 0 - 5 mol/L concentration. As the amount of displaced contaminants did not increase on further additions of DETA, it is apparent that they were not originating from the stock solution of the additive. This fact also suggests that complete displacements of the detected contaminants were achieved. These findings led us to apply DETA also in the cleaning procedure for sample containers (see procedure B in the Experimental Section). In spite of the fact that none of the sample contaminants migrated in the positions of the analytes and the use of a more selective detection wavelength ( 3 10 nm) did not visualize them (Figure 5d), a thorough cleaning of the sample containers by this procedure was always employed. The above facts also imply that similar displacement effects can be induced by some sample constituents with apparent analytical consequences, especially, for trace analytes. Undoubtedly, the use of microvessels with a less favorable surface/ volume ratio, as used for sample storage in CZE, can further increase this risk. Analytical Performance and Application Potentialities. At present there is no generally accepted procedure for estimating the limit of detection (LOD) in CZE. However, a close Analyfical Chemistry, Vol. 66, No. 7 7, June 1, 1994
1821
Table 3. Limits of Detection for Paraquat and Diquat in Tap Water at 310 nm' constituent
Table 4. Reproduclbliitles of the Determinations of ppt Concentratlons of Paraquat and Diquat In Tap Water' sample no.
parameter
paraquat
diquat
constituent
N,, (AU) S (AUifmol) LOD (fmol)
2.0 x 10-4 18.9 x 10-7 64
2.0 x 10-4 18.0 x lo--
paraquat
67
a The slopes of the calibration curves (Sj were obtained by linear regression ( r = 0.9990) for 1-10-pmol amounts (eight data points) of the herbicides in tap water. The peak-to-peak noise (Npp) wab obtained in the same experiments for 30-s regions of the base line on the electropherograms. The measurements were carried out in the electrolyte systems ITP-1 and ZE-la.
analogy of Z E with elution chromatography makes the use of procedures recommended for chromatography justifiable. We employed the IUPAC model, conveniently modified for chromatography,jj and eq 1 was used for the calculations of actual LOD values, where N,, is the peak-to-peak noise and S is the analytical sensitivity (slope). The relevant data for
the herbicides are summarized in Table 3. Our LOD values for both paraquat and diquat are 3-4 times higher than those reported by others.2 Uncertainties in the contents of water in the preparatives of the herbicides (see, e.g., refs 40-42) can provide only partial explanations for these differences. As information concerning the procedure employed in the estimations of the LOD is limited in the quoted work, it can be only speculated that the lower LOD values are associated with the use of electrokinetic injection.j6 Minimum concentrations of the analytes which can be determined by the ITP-CZE tandem are important performance characteristics when its use in trace analysis is considered. The LOD values give only partial information i n this respect as a maximum sample volume which can be taken for the analysis may play a key role. The load capacity of the ITP stage determines this parameter, and its relations to the sample composition are straightforward.18 We found experimentally that the use of the ITP stage enables injection of tap water samples as large as 90 FL. This is a 103-fold increase in comparison to the maximum sample volume which can be directly injected into the same CZE column under identical working conditions. Benefits of the ITP-CZE tandem in the analysis of paraquat and diquat in water are illustrated by electropherograms in Figure 6. Two tap water samples spiked with the pesticides at ppt concentrations were analyzed. From the electropherograms and from the corresponding data (Table 4) we can see that 2 X 10-9-6.8 X 10-9mol/Lconcentrationsoftheseanalytes could be determined with good reproducibility. A high analytical selectivity is also clear from these electropherograms. A higher concentration of h a + ions in the other type of sample taken in this study (Lake Ontario water) reduced proportionally the above maximum injection volume to ca. 60 yL, and obviously, the minimum detectable concentrations (55) Foky, J. P.; Done), J. G . Chromatographia 1984. 18. 503-51 I (56)Jackson, P. E.; Haddad, P. R . J Chromarogr. 1993. 640. 381-487
1822
Analytical Chemistry, Vol. 66, No. 11, June
I, 1994
parameter*
1
2
(PL) c (nmoliL) n (fmol) h (ACT)
25 6.78 169.5 3.8 X 10" 0.2 x 10" 25 5.10 127.5 2.7 x 10-4 0.2 x 1 0 4
85 2.68 227.8 5.0 x IO"' 0.4 x 10-4 85 2.02 171.7 4.0 X IOd 0.2 x 10-4
JJ,"]
sh
diquat
(.4U)
(rL) c (nmol/L) n (fmol) h (AI;) L,"]
(h
(AC,
Tap water samples spiked with the herbicides were analyzed five times within 3 days in the electrolyte systems ITP-1 and ZE-la (Table 1) with the photometric detector set at 310 nm. Vinj = injection volume; c = concentration of the analyte; h = mean value of the peak height; s h = standard deviation for the peak height.
*
increased in the same proportions. This agrees with the fact that the relative differences in the effective mobilities of the herbicides and Na+ ions determined their resolution rates in spite of the fact that the sampling ratio4*was favorable. From illustrative electropherograms in Figure 7 we can see that a high analytical selectivity was achieved in the CZE stage. When the concentrations of the herbicides which could be quantified in tap water (Figure 6) and the minimum detectable concentrations estimated from the LOD values (Table 3) for an 85-pL sample injection volume are compared to the minimum detectable concentrations reported for CZE,2it is apparent that the ITP--CZE tandem offers a 103-fold improvement. A comparison to the ITP analysis with conductivity detection8,'" suggests that this represents an improvement in the range of 104-105 for similar injection volumes when the precolumn sample preparation in ITP is ignored. Also a comparison with the data reported for a very sensitive HPLC assay42indicates that our capillary electrophoresis approach can provide, for the same sample volumes. approximately 1O2 lower minimum detectable concentrations. The ratios of the mean values of the peak heights of the herbicides for the tap water samples agreed, within random errors, with the values calculated from the injected amounts (Table 4). This implies that nosignificant losses oftheanalytes occurred at 2-7 X mol/L concentrations. The ITPC Z E tandem should be a convenient alternative to the analysis of even lower concentrations of paraquat and diquat in water. However, an increase of the load capacity of the ITP stage is an essential requirement in achieving this goal, especially, for samples containing the matrix constituents (e.g., h a + , K + . Ca'+ , ME:+) a t higher concentrations. Although this can be achieved in a well-defined way.4x there is an inherent limit when the ITP-CZE tandem is considered. This limit is set by the load capacity of the CZE stage itself and by the minimum size of the sample fraction which guarantees a complete recovery of the analyte i n its ITP-CZE transfer. Electropherograms in Figure 8 show that resolution problems due to overloading of the C Z E column can occur in such critical situations. The same samples of tap water as used i n experiments in Figure 6b were used for the analyses. The only difference was in the amount of transferred N n + ions
*
,pD
II * +
P I D
PD
i
55
:
295
moi/L and Flgure 8. Separation of paraquat and diquat in tap water at ppt concentrations. (a) The sample contained paraquat at 6.8 X moi/L, and the injection volume was 25 pL: (1) tap water spiked with mol/L DETA (blank run), (2) tap water immedlately diquat at 5.1 X after the spiking with the herbicides, (3) run ca. 20 min after the spiking, (4) run ca. 30 h after the spiking. (b) The sample contained paraquat moi/L and diquat at 2.0 X lo-0 mol/L, the concentration of DETA was moilL, and the injection volume was 85 pL (25-pL valve at 2.7 X 60-pL microsyringe): (1-4) the same meaning as for sample a. The separations were carried out in the electrolyte systems ITP-1 and ZE-la with photometric detection at 310 nm and with driving currents 250 and 200 pA in the ITP and CZE stages, respectively. The time for the ITP preseparation was 6 min.
+
0
P Tris
i
D
*,**
DETA \
- 20 \ 9
\
t
P
145
265
145
265
145
265
16) Figure 7. Separation of paraquat and diquat in Lake Ontario water: (a) water sample containing moi/L DETA; (b) water sample as in moi/L) and diquat (1.6 X part a spiked with paraquat (2.1 X mol/L); (c) water sample as in part a spiked with paraquat (13.0 X mol/L) and diquat (9.8 X mol/L). The sample injection volumes were 60pL. The separation was carried out in the electrolyte systems ITP-1and ZE-lb with photometric detection at 310 nm and with driving currents 250 and 200 pA in the ITP and CZE stages, respectively. (*, * ) = migration positions of paraquat and diquat, respectively. The time for the ITP preseparation was 6 min.
135
255
375
t (9 Figure 8. Overloading of the CZE column with Na+ ions. The same tap water samples (85-pL injection volume) as in Figure 6b were separated except that the DETA concentrations were increased to lo-' mol/L. The transferred fraction is marked on the Na+ zone with a dashed ilne. (a) Unspiked tap water. (b) Tap water spiked with paraquat (2.7 X moi/L) and diquat (2.0 X moi/L). The separation was carried out in the electrolyte systems ITP-1 and ZE-1b with photometric detection at 310 nm and with driving currents 250 and 200 pA in the ITP and CZE stages, respectively. ( , *) = migration positions of paraquat (P) and diquat (D), respectively. The time for the ITP preseparation was 6 min.
into the C Z E stage (approximately 10 times more than in the a rapid screening for a maximum expected concentration of runs in Figure 6 ) . This column overloading is manifested via the ITP migration of the analytes in the CZE stage in a way the analytes in the samples could be performed in this way. already described in detail for a single-column s i t u a t i ~ n . ~ ~ , ~ ~ Approximate calculations based on the electropherograms in CONCLUSIONS Figure 8 show that the peak obtained in the run with 85 pL The coupled-column ITP-CZE technique provides an analytical potential for environmental trace analysis of of unspiked tap water corresponds to an 8 X 10-lo mol/L ionogenic herbicides such as diquat and paraquat and other concentration of the herbicides. However, it must be noted polar pollutants. This technique reduces the minimum that this peak covers all detectable constituents focused by detectable concentration of ionogenic solutes by a factor of ITP between theNa+ and Tris+ zones. Although identification 103-104 when compared to single-column CZE. This sigcertainty was reduced by this focusing effect, it is clear that Analytikal Chemistry, Vol. 66,No. 11, June 1, 1994
1023
nificant improvement is related to an increased sample injection volume as the ITP stage provides a favorable enrichment factor due to the ITP preconcentration combined with a well-defined sample cleanup. The limit of detection for paraquat anddiquat is in the mol/L concentration range, which makes this technique suitable for trace analysis of these herbicides in aquatic matrices. Sample preconcentration techniques based on liquidliquid8.10and solid-phase e ~ t r a c t i o n ~techniques '~~* coupled with the ITP-CZE tandem appear to be promising, in the near future, new developments for separating low and high molecular weight solutes. Such a combination will decrease the minimum detectable concentration of solutes to low ppt
1024
Analytical Chemistry, Val. 66,No. 11, June 1, 1994
or even ppq levels without using sophisticated and expensive detection systems. ACKNOWLEDGMENT D.K. thanks the National Water Research Institute, Burlington, Ontario, for a fellowship. This work was also supported by a grant provided by Slovak Grant Agency for Science under b o . 1 /990901/92. Received for review November 2, 1993. Accepted March 8, 1994.@ *Abstract published in Adoonce ACS Abstracts, 4prii 1 5 ~1994
