Anal.
3726
chsm.1993, 65, 3726-3729
Large Volume Stacklng of Anions in Capillary Electrophoresls Uslng an Electroosmotlc Flow ModHler as a Pump Dean 5. BUM Genomyx Corporation, 460 Point San Bruno Boulevard, South San Francisco, California 94080
INTRODUCTION In the last few years, it has been shown that free-solution capillary electrophoresis (CE)can be used to concentrate samples.14 Sample concentrating can be achieved by either isotachophoretic or field-amplified methods. The former techniques use either a trailing electrolyte1 or a leading electrolyte2added to the sample plug. The sample then stacks itself into the column on the basis of ita ion mobility relative to that of the additive. The field-amplifiedmethods rely on the differencesbetween the concentrations of the sample region and the run buffer region. The sample region generates a higher applied electric field; thus the velocities of the analyte are greater than those in the run buffer region. The sample will move rapidly to the concentration boundary, and then will slow down and stack itaelf.3 One method, field-amplified sample injection, can concentrate material up to 1000-fold over conventional electrokinetic injection; however, there is a bias associated with the method? The hydrodynamic method, large volume sample stacking using polarity switching,has also been shown to concentrate material up to 85-fold over conventional hydrodynamic inje~tion.~ The polarity switching allowsthe large water plug left after sample stacking to be removed. The removal of the large water plug is needed bacause the water w i l l perturb the electric fields during separation of the analyte. A bias associatedwith this method occurs when the injectionvolume to column volume ratio exceeds that of the electroomotic flow to ion mobility ratio.6 Also, switching the polarity can lead to irreproducible results if the current is not monitored correctly. Here, a scheme is presented for largevolume sample stacking for negative species that avoids switching of the polarity to pump the water out of the system.
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1
All experiments were performed on a SpectraPhoresis 1OOO.
All equipment control and data handling were performed on an
IBM Model 80 with OS2 as an operating system ueing SpectraPhoresissofware. The run buffer consisted of 3 mM pyromellitic acid (PMA),3 mM NaOH, and 1mMdiethylenetriamine(DETA). The anion standard consisted of 10ppm of chloride (Cl-1, nitrate (NOS-),sulfate (SO&, and oxalate (OX-) prepared in water and then diluted to the needed concentrations. All chemicals were purchased from Aldrich and prepared in MilliQ 18 Ma water, The run conditions were as follows: untreated fueed silica capillaries 50 pm x 45 cm and 100 pm X 45 cm with detector windows burned at 32 cm;separation voltages -20 kV,5 mA for the S p m column and -20 kV,22 mA for the 100-pm column; detection wavelength 254 nm with a 1-s time constant; run temperature 20 "C. Since the separations were performed at 20 OC, the NOS-and SO2-peaks coelute. Injectionswere performed (1)Jandik, P.;Jones, W. R. J. Chromatogr. 1991,546,431-443. (2) Gebauer, P.;Thormann, W.; Bocek, P. J. Chromatogr. 1992,602, 47-57. (3) Burgi, D. S.; Chien, R.-L. Anal. Chem. 1991,63, 2042-2047. (4)Chien, R.-L.;Burgi, D. S. J. Chromatogr. 1991,559,153-161. (5)Burgi, D. S.;Chien, R.-L. Anal. Biochem. 1992,2,308-309. (6)Chien, R.-L.;Burgi, D. S. Anal. Chem. 1992,64,489A-496A. 0003-2700/93/0365-3726$04.00/0
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H20
Q
samplb
'sample'
H20
0
Buffer
hydrodynamic injection
Fig l a -30kV
4-
anion mobility
Buffer
1
high voltage is applied ions start to slack up at the concentration boundary
-3OkV
,
ground
uffer
Fig 1b
, 4-um
:II kI Buffer
-
Fig IC
ground
Buffer
H20
the water is being pumped out ions are stacked at concentration boundary
-30kV
ground anion mobility
Buffer
Fig Id
EXPERIMENTAL SECTION
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4 Buffer
the water is completely pumped out ion separation occurs
Flguro 1. A schematic diagram of how the water pumps itdl out of the column: (a) once the sample Is InJectedInto the column, the DETA ontheceplrvywaW~I~thewaterofthesampbp~lnaeedng the ( potmtlal in the sample r w n ; (b and c) after the vottage Is applkd, the ions stay at the boundary until the water plug Is pumped out of the column; (d) the water Is completely pumped out of the column; (d) the water Is completely pumped out of the column, and the anlons separate under normal CE condltkns.
under vacuum for 3 and 30 s at low vacuum which have a % RSD of injection of 2.3 and 24 8 and 60 8 at high vacuum. The 24s injection half fiied the column and the 60-8 injection completely filled the column.
RESULTS AND DISCUSSION To perform large volume sample stacking, the electrophoretic mobility of the ions of interest must be negative or opposite with respect to the electroosmoticflow. In polarity switching large volume stacking, the configuration of the electrodes are switched after the sample is loaded into the column. Once the sample buffer plug is removed, the electrodes are switched back to their original configuration and separation of the ions occurs. In large volume sample (B 1893 American Chemical Sockty
ANALYTICAL CHEMISTRY, VOL. 65, NO. 24, DECEMBER 15, 1993
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1 Oppm standard 3sec injection
-0.0001
-0.0002
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-0.0005
N03-\l
-
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1 ppm standard
30sec injection
Migration time (min) I
2.'90
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3 .'lo
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Flgun 2. A comparison of a 3-s hyaodVnamic lnjactbn of 10 ppm of a n h s with a 30-8 injection of 1 ppm of
1 ppm standard
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l.'Ol
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3.50
anions In a 50-pm4.d. column.
30sec injection
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Migration time (min) I
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Flguro 8. An ekctrophewwam of the 30-8 Injection including the current
trace.
stacking with an electroosmotic flow modifier, the electrode confiiation is not changed after sample loading. The negative speciee move toward the ground electrode when -20 kV is placed on the injection end of the column. Figure 1is a schematic diagram of how the water pumps itself out of the column. In conventional ion analysis, a concentrationof 1mM of DETA in the run buffer suppresses the eledrooemotic flow by reducing the f potential on the column wall.' The electroosmotic flow is so slow usingDETA that after a 3-h run at -30 kV the neutral marker is not detected. Also no evidencewas found concerningthe reversal of the electroosmotic flow at higher DETA concentration during theae experiments. More studies on the effecta of concentration of DETA are ongoing. In large volume sample stacking once the large volume of sample is introduced into the column, the DETA on the capillary wall diesolves into the water of the sample plug increasing the f potential in the sample region (Figure la). Next the voltage is applied, and the local electroosmotic flow
generated by the sample region w i l l move the bulk solution toward the anode of the system (Figure lb). At the same time, the negative ions in the sample r e o n will stack themselvea up against the boundary between the sample region and the run buffer The ions will stay at the boundary until the water plug is pumped out of the column because almost all of the applied electric field k+opped across the water plug (Figure IC).Once the water leaves the column, the applied field is dropped across the run buffer region (which is now the whole column) and the ions begin to separate again (Figure Id). The run buffer region that is pulled up into the column from the reservoirs contains DETA which again coats the column wall and suppressea the f potential. Figure 2 is a comparison of a 3-8 hydrodynamic injection of 10 ppm of anions with a 30-8 injection of 1ppm of anions. The difference in migration times between the two runs is the amount of time it takes the water plug to pump itself out ofthecolumn (-10s). The twoeledropherogramsareeimilar in peak shape, peak area, and ion mobility, which indicates that the water plug is completely removed from the column.
(7) Kelly, L.;Nelson, R.J. J. Liquid Chmmatogr.l993,16(9 and 101, 2103-2112.
9721)
ANALYTICAL CHEMISTRY, VOL. 65, NO. 24, DECEMBER 15, 1993 1OOppb standard half-filled column
0.0001
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Migration time (min) -0.0004
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3 .I30
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E-gram Analysis: \PUMP5.8FF A U X File: \PUMPS.BFF
Flgvr 4. Comparison of a 3-
InJectIon of 100 ppb of anion with a half-fllled column Injection In a 50-fim column.
0-
1
Current trace
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Migration time (min)
1.I20
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Figure 3 is the complete electropherogramof the 30-8injection of the 1 ppm sample. During the f i t 30 s, the water plug isbeing pumped out of the column, as indicated by the current trace increasing until it plateaus (thefiit 10s is the reaponae time of the HV power supply controller). When the current trace plateaus, the water plug is completely removed from the column and the current trace will remain stable for the rest of the separation. For both the 3- and 30-8injection of the %RSD falls within the instrument's ability to inject reproducible volumes. The migration timea of the ions have a %RSD of 1.7 which indicates the water is completely removed from the column. Figure 4 compares a 30-8 injection of 100 ppb of anion VB a half-fiied column injection. The resolution of the analysis is preserved even at these large volume injections. The reproducibility of the half-fiied injection was not studied since it is used only to show that the water plug can be removed after a large volume of sample buffer is injected. Figure 4 shows a &fold increase in peak height of the half-fiied column over the 30-8 injection which is supported by the calculated volumes of 870 and 180 nL,
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,
4 .I80
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6.00
respectively. The difference in migration times isthe amount of time it takes for the water to be pumped out of the column. Figure 5 is an electropherogram of a whole column fiied with 10 ppb of the ion standard. The water is pumped out of the column using the DETA pump. In Figure 5, some of the OX-is lost because this slow-movingion is pumped out by the faster electroosmotic flow, but the detection of 10 ppb of C1- and NOa- is achieved with the whole column stacking. It took over 2.5 min to pump the water out of the column at an average electroosmotic flow of 5.7 X lo-' cm2/(Vs). Figure 6 is a comparison of a 10 ppb concentration of the anion standard for a 6-vs, 30-8injection using a 100-pm4.d. column. The peak heights are about 5 times larger for the 30-over the 6-8 injection. The baseline is not completely stable, possibly due to thermal effects for the current being 4 times higher than for the 50-fim column. Also with the 6-8 injection, the temperature is high enough for the ion mobility of the NOS-to separate it from the SO4%.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 24, MCEMBER 15, lQ93 3728
n
0.0002
lOppb standard 6sec injection
-0 * 0002
-0.0006
ox-
-0.0010
I Oppb standard 30sec injection -0.0014
H03-,s04-q
Migration time (min)
-0.0018 2
Flgure 6. A
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2
.bo
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3 .'lo
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3 .I70
4 .loo
comparison of a 10 ppb concentration of the anion standard for a 8-s vs 30-s injection uslng a 100-wn4.d.column.
CONCLUSION Evidence has shown that a system which has a dynamic coating for suppression of the electroosmoticflow can be used to concentrate negative species. A 100-foldimprovement of detection of the fast-movingions is achieved by whole column stacking. The 100-pm column data indicates that CE can be used as a microsample preparatory system. Investigation into large volume concentrating of positive ions with the proper choice of coating and electric field configurations is ongoing.
ACKNOWLEDGMENT This work was performed at SpectraPhysic Analytical in Fremont, CA. 1would like to thank Dr. Lenore Kelly for her support and conversations on this work.
RECEIVED for review May 1993.
4, 1993. Accepted October 1,
