Anal. Chem. 1994,66, 2578-2583
Auxiliary Electroosmotic Flow for Postcapillary Reaction Detection in Capillary Electrophoresis Rlchard M. Cassldy,' Wenzhe L u , ~and Val-Pul Tse Chemistry Department, University of Saskatchewan, Saskatoon, Saskatchewan S7N 0 WO, Canada
The application of a supplementary potential at the end of a separation capillary has been evaluated for the introductionof a reagent solutionfor postcapillaryreactiondetection. Analyte zones emerging from the capillary were introduced into a short ( -2 cm) reactioncapillary that was alignedwith the separation capillary. The efficiency of transfer of the analyte zones to the reaction capillary and the relative flow of reagent solution and separation electrolyte into the reaction capillary were a function of the applied potential at the intersection of the two capillaries. Capillaries having i.d. values of 75,50, and 25 pm were investigated. Whena 2 cm X 25 pm i.d. reaction capillary was added to a 60 cm X 25 pm i.d. separation capillary, the changein peak dispersion for the transfer of nitrite peaks across the gap and through the reaction capillary was small (decrease from 420 000 to 400 000 theoretical plates) when the percent flow from the reagent reservoir was 30%of the total combined flow. The influenceof displacement between the capillaries on peak transfer was similar for both axial and vertical directions over the range of 1-50 bm. A potential application examined for this detectionsystem was the additionof a 0.4 mol/L strong base solution to permit the electrochemical detection of carbohydrates separated in a borate buffer system. Capillary electrophoresis (CE) has undergone rapid development in the last few years as an analytical separation procedure, but detector limitations still restrict the range of application of this efficient separation technology. Although impressive results have been shown for fluorescencedetection,' limited sample volumes result in poor sensitivity for UVvisible absorbance detection. Electrochemical detection methods are much more suited to the small sample requirements of CE,2 and this characteristic may result in greater application of electrochemical detection in CE than has been the case for liquid chromatography (LC). However, there is still a need to expand the range of compounds that can be analyzed by existing CE detector technology. Successful development of on-line postcapillary reaction detection (PCRD), which has played a major role in the development of LC, would improve the sensitivity and range of application of CE detectors. In spite of the obvious difficulties associated with interfacing small internal diameter capillaries without loss of separation efficiency, some limited studies have been reported in this area. Early applications relied on pneumatic f Present address: Department of Chemistry, University of New Orleans, New Orleans. LA 70148. (1) Arriaga. E.; Chen, D. Y.; Cheng, X. L.; Dovichi, N. J. J. Chromarogr. 1993, 652, 347-354. (2) Curry, P. D., Jr.; Engstrom-Silverrman, C. E.; Ewing, A. G. Electroanalysis 1991, 3, 587-596.
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introduction of the reagent solution,34 an approach that has been successful in LC. Unfortunately such approaches can distort peak profiles in CE, and efficiencies reported were low (320 Vcm-l. When the PCRD was left floating at zero applied voltage, there was very little change in migration rates because the separation voltage caused uniform EOF in both the separation and PCRD capillaries. All capillary systems showed a slight increase in migration times with increased gap distance, with the percentage increase being 25 >> 50 > 75 pm. Curves A-D in Figure 2 show the increases observed for 25-pm systems having PCRD gaps from 1 to 20 pm. The percent change in migration time for the different systems studied was 16% for 25 pm (1-20 pm gap), 3% for 50 pm (1-60 pm gap), and
ZT cp 0 0
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PCR Potential (V) Figure 3. Change in peak height for catechol as a function of PCRD potential. Experimental conditions are as for Figure 2; axial PCRD gap distances are as indicated: 25 pm i.d. capillaries.
systems. Both peak height and peak areas (not shown) declined above PCRD voltages of 600 V because of dilution with increasinginflow of PCRD electrolyte. Changes in peak height and area for 50 and 75 pm i.d. capillaries were obscured at high PCRD voltages because of shifts in the potential field at the working electrode. Consequently, to reduce the effects of PCRD voltage 25-pm capillaries were used for all further studies. The above results also showed that a grounded PCRD gave poor performance, and all further tests at zero applied PCRD voltages maintained the PCRD at a floating potential where the zero potential was determined by the separation potential. It should also be noted that the experimental arrangement for a grounded PCRD is similar to the conditions present in “off-column” detection recommended to reduce noise in electrochemical detection.18J9 The disadvantages of such approaches are obvious. Although the use of a balanced back-pressurecan reduce losses in efficiency due to parabolic this is not an attractive experimental approach for routine applications. The number of theoretical plates obtained with catechol and cadmium with PCRD systems was only in the range of 50 000-1 30 000, and these efficiencies were essentially the same as those obtained in the absence of a PCRD system. While this efficiency was suitable for initial evaluation, narrower peaks were required for more critical evaluation of peak dispersion. Consequently, nitrite was used as a test analyte because it exhibited efficienciesof 420 000 theoretical plates (20-kV separation voltage), which is close to that expected theoretically if diffusion is the major source of band broadening. This feature combined with migration in the same direction as the EOF gave small-volumepeaks and, thus, made nitrite a sensitive probe for peak dispersion effects in the PCRD. Plate numbers for nitrite (corrected for sampling variance, see ExperimentalSection) were measured for PCRD gap displacements from 1 to 50 pm. The results in Figure 4 for axial displacements of 1 and 10 pm (curves C and B) show that both of these experimental arrangements gave good efficiency as long as some additional EOF (PCRD voltage > -600 V) was introduced in the PCRD capillary; it should be (18) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1987, 59, 1762-1766. (19) Kok,W. T. Presentationat Fifth InternationalSymposiumonHigh Performance Capillary Electrophoresis, Orlando, FL, 1993.
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_
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200 400 600 800 1000 Applied PCR Voltage ( 4 )
Figure 4. Effect of postcapillary reaction potential on Separation efficiency and peak height for 25 pm i.d. capillary. Experimental conditions: 2 X lo-‘ mol/L nitrite sampled at 1 kV for 5 s; electrolyte, 0.005 mol/L phosphate and 0.005 mol/L cetyltrimethylammonium chloride electrolyte at pH 6.5; separationand PCRD capillariesare 60 and 2.2 cm, respectively; separation voltage, -20 kV; curves A and C, 1-pm gap; curves B and D, 10- and 40-pm gaps, respectively.
noted that a nominal displacement of 1 pm is likely equivalent to 1-3 pm due to experimental errors and variations in the angle of capillary cleavage. Plate counts (400 000) at a PCRD voltage of -1000 V were close to the maximum observed for the separation capillary alone (420 000). Peak heights for a 1-pm displacement (curve A, Figure 4) showed an initial increase due to improvements in efficiency, followed by a leveling off due to dilution from electrolyte injected at the PCRD joint. At low PCRD voltages, the efficiency for a 1-pm displacement was lower than that for a 10-pm gap, possibly due to the importance of capillary alignment in a vertical direction when the applied PCRD voltage was small. Results obtained as a function of PCRD voltage at larger displacements showed a similar trend to that for the 10-pm displacement, but with an overall gradual shift to lower efficiencies as illustrated for the results in curve D, Figure 4, for a 40-pm displacement. The results in Figure 4 suggested that capillary alignment in vertical directionsinfluenced separation efficiencyfor small PCRD voltages. However, it was not clear what effect small displacements would have when the PCRD voltage was adjusted to inject PCRD electrolyte, a condition required for PCRD detection. Since measured peak variance (time units) should be a maximum when the injection of PCRD electrolyte is relatively slow, a PCRD voltage of -800 V was chosen for this evaluation. The effect of small displacements on separation efficiency was studied for axial, vertical (upwards and downwards), and horizontal (sideways) directions; the latter three directions are essentially equivalent but were studied to ensure that irregularities in the fractured surfaces of the two capillaries did not bias the results for any one direction. Figure 5 shows the results for this study. For separations in downwards, upwards, and sideways directions, an axial separation of 1 pm was always maintained between the two capillaries. Each direction of displacement was studied over the entire displacement range, and then the capillary was repositioned at a 1-pm axial displacement for the next series of measurements. Consequently, the range of values of theoretical plates at 1-pm displacement in Figure 5 represents AnalyticalChemistry, Vol. 66, No. 15, August 1, 1994
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Flgure 5. Variation of separatlon efflclency with axial and vertical dlsplacementsat PCRD. Experimentalconditkns: PCRD potential,-800 V; other conditlons as for Flgure 4.
the range of experimental errors expected for alignment of the two Capillaries. The number of theoretical plates observed at the PCRD voltage used (-800 V) is in agreement with previous data obtained for a study of separation efficiency as a function of applied PCRD voltage, which was shown in Figure 4. It is interesting to note that the results in Figure 5 suggest that a given increase in the size of the axial gap between the two capillaries is equivalent to the same displacement in any of the other directions where the axial gap is always 1 pm; this was true even for relatively large displacements of 50 pm. Apparently, the potential fields between the capillaries maintain a uniform flow for all configurations. Since these results suggest that vertical displacement may not have an inordinately large effect on peak dispersion, the design of holders for PCRD capillaries may not be as formidable a task as it might initially seem. The rate of electrolyte injection at the PCRD gap was calculated from results obtained from measurements of detector response as a function of PCRD voltage for three sets of conditions: analyte placed in both the separation and PCRD electrolytes; analyte placed in only the PCRD electrolyte; and only electrolyte in separation and PCRD capillaries (for measurement of background signal as a function of PCRD voltage). The relationship between applied voltage and calculated rate of flow from the PCRD is shown in Figure 6 . For a -20-kV separation voltage, the voltage across a 2.2-cm PCRD capillary attached to a 60-cm separation capillary would be -707 V if the junction potential is allowed to float. To achieve a PCRD injection rate equal to the EOF in the separation capillary, the potential field in the PCRD capillary would have to be double that in the separation capillary ( V E O F = ~ E O F Ewhere , VEOF is the rate of EOF, ~ E O Fis the electroosmotic mobility coefficient, and E is the electric field strength). Thedatain Figure6 showthat a 1:l flow isobtained at a PCRD voltage of -1400 V, in excellent agreement with what is expected (2 X -707 V). The small flow (7%) observed at zero applied volts is due to experimental error. To further evaluate this PCRD system, the possibility of injection of a strong base to enhance electrochemical detection of carbohydrates, as is done in liquid chromatographic procedures, was examined briefly. Carbohydrates have been separated in CE as their borate complexes2G22and in strong 2582
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PCR Voltage (-V) Flguro 6. Flow of PCRD electrolyte as a functlon of appiled PCRD voltage. Experlmental condHlons are similar to those in Figure 4; the experlmental procedure used to calculate flow was described In the text.
ba~e.23-~~ Borate electrolytes offer alternate selectivity patterns and can be used in larger i.d. capillaries, whereas the 0.1 mol/L hydroxide electrolytes required for carbohydrate ionization must be used with 10-pm i.d. capillaries if 20-30kV separation voltages are used.23 Unfortunately, borate decreases the response of copper and gold electrode^,^^*^^ both of which offer sensitive electrochemical detection in a strong base without any requirement for derivatization. Recent studies in our laboratories have shown that in the presence of large concentrations (>O. 1 mol/L) of hydroxide, the response can be improved at copper and gold electrodes. Since copper electrodes do not require a pulsed voltage scheme for detection, it was chosen for these initial evaluations. When the PCRD system was used to inject 0.4 mol/L sodium hydroxide into a borate electrolyte, electrode response was not observed for any of the test carbohydrates. Subsequent studies showed that this lack of response was a result of the migration of hydroxide through the separation capillary toward the positive separation voltage electrode (against the EOF) rather than into the PCRD capillary. Such counter-migration of reagent ions is important no matter what mechanism is used to introduce a PCRD reagent into a CE system, and proper consideration of this effect is required both to avoid chemical changes in the separation capillary and to ensure that the reagent does move toward the injection point. To avoid counter-migration of hydroxide, theseparation potential was reversed, and thus reversal of the EOF was necessary to permit migration of the carbohydrates to the detector. Cationic surfactants, commonly used to reverse EOF, destroyed detector response, but good response was obtained in the presence of the cationic polyelectrolyte, poly( 1,ldimethyl-3,5-dimethylenepiperidiniumchloride) (PDDP) .The poorer performance of the surfactant systems may be due to the formation of hydrophobic monolayersor bilayer structures, which could interfere with the diffusion of charged carbohydrate species to the electrode. PDDP provides stable ~
(20) Hoffstettcr-Kuhn, S.;Paulus, A.; Gassmann, E.; Widmcr, H. M. Anal. Chem. 1991,63, 1541-1547. (21) Honda, S.;Iwasc, S.;Makino, A.; Fujuwara, S . Anal. Biochem. 1989, 176, 72-17. (22) Lui. J.; Shirota, 0.; Novotny, M. A n d . Chem. 1991, 63, 413417. (23) Lu, W.; Cassidy, R. M. Anal. Chem. 1993,65, 2878-2881. (24) Colon, L. A.; Dadoo, R.;Zarc, R. N. Anal. Chem. 1993,65, 476-481. (25) O’Shea, T. J.; Lunte,S. M.; LaCoursc, W. R. Anal. Chem. 1993.65.948-951,
7 for a PCRD voltage of zero (no injection of base) was obtained at a sensitivity setting 70 times that used to display thedetection of thecarbohydrates. At this increased sensitivity level, only one peak (inositol) was observed, and its peak height was equivalent to the peak-to-peak baseline noise ( 5 PA). Maximum separation efficiency was observed for maltose, which gave 110 000 plates. The results from peak dispersion studies discussed above show that band broadening properties of the PCRD should not be a limiting factor in the efficiency of this separation. Indeed, efficiencies of 100 000 plates20 and lessz1 appear to be characteristic of separations of carbohydrates as borate complexes; separations in basic solutions give 100 000-200 000 theoretical p l a t e ~ . ~ Sen3*~~ sitivities, as measured by peak current, were about 10 times 0 100 200 300 400 500 600 700 less than that observed with the more efficient base electrolyte Time (s) but further optimization, such as the use a gold electrode in a pulsed mode, and/or displacement of the Figure 7. Separation and detection of carbohydrates after injection of 0.4 mol/L sodium hydroxide at PCRD. Experimental conditlons: electrode from the end of the PCRD capillary should result separation electrolyte, 0.05 mol/L borate with 0.2% (w/v) PDDP at pH in improved signal-to-noise ratio without sacrificing separation 9.5;PCRD electrolyte, 0.4 mol/L sodlum hydroxide; PCRD gap, 10pm; efficiency of the carbohydrates. separation and PCRD voltage, -15 kV and -800 V, respectively: detection at 25-pm copper disk electrode: sample concentrations, 2 The results in Figure 7 and other data presented above X lo4 to 2 X lo3 mol/L. Peak identity: 1, inositol; 2, glucose; 3, suggest that electroosmotic control may offer advantages over sorbitol; 4,mannose: 5, rhamnose; 6, maltose; 7, 0-cyclodextrin. previously reported methods for reagent addition in postcapillary reaction CE detection, particularly with regard to reversed EOF, even at low concentrations (>0.03% w / v ) , ~ ~ reducing band broadening processes in the PCRD. The operation of these PCRD devices does not appear to be difficult, and thus faster analysis of easily resolved carbohydrates was and due to rapid diffusional mixing, a 2-cm reaction capillary possible; the pH dependence of the EOF is also eliminated in will provide a longer reaction time than that normally provided the pH range of 6.5-10? In addition, larger carbohydrates, by low dead volume reactors used in LC; the time required such as j3- and y-cyclodextrin and maltoheptaose, which did for diffusion from the center of a 25-pm capillary to the wall not migrate from the capillary when hydroxide electrolytes should be of the order of 0.08 s . ~ The major difficulty were used, could be separated and detected in the presence encountered in these studies was the formation of bubbles of this polyelectrolyte; analyte sorption onto the capillary has when the PCRD reactor was first assembled; bubbles formed also been proposed as a cause of a time-dependent loss of at this time seemed to be difficult to dislodge. Once in use, resolution in borate electrolytes.21 Concentrations of PDDP however, bubble formation was not a problem because under of up to 3% (w/v) in strong base electrolytes produced some these conditions the bubbles were easily transported through changes in carbohydrate electrophoreticmobility, but changes the PCRD capillary. The problem with initial bubble in relative migration rates were small. More significant formation may be solved with the use of degassed electrolytes, changes in selectivity may be possible in borate systems where but this was not explored in these studies. However, it is clear larger charges may exist on the carbohydrates, but this was that additional studies are needed to explore such factors as not examined. long-term flow stability, theeffect of ionic strength differences, Figure 7 shows that in the absence of an applied PCRD PCRD interface design, and the applicability of this approach voltage no carbohydrate peaks were visible with the borate/ to practical analytical situations where slow reaction rates PDDP electrolyte, but when the application of -800 V across may limit detector response. the PCRD capillary was used to inject 0.4 mol/L sodium hydroxide, it was possible to detect all of the seven carbohydrates present in the sample. The signal shown in Figure Received for review January 3, 1994. Accepted May 6, 1994." I
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(26) Stathakis, C.; Cassidy, R . M. Anal. Chem., in press.
Abstract published in Aduonce ACS Absrrucrs. June 15, 1994
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