Device and method for separation and analysis of trace and ultra-trace ionogenic compounds by isotachophoresis and zone electrophoresis on chip
10564121 ยท 2020-02-18
Inventors
Cpc classification
G01N27/44773
PHYSICS
International classification
Abstract
Disclosed herein is a device for separation and analysis of trace and ultra-trace ionogenic compounds by isotachophoresis-zone electrophoresis on a chip with online detection and method of its use, concentrating trace and ultra-trace analytes by isotachophoretic migration in a wide separation channel when a manifold of auxiliary electrodes is employed. Then the isotachophoretic zones of trace and ultra-trace analytes are transferred by isotachophoretic migration through a tapered channel, while corresponding auxiliary electrodes are progressively disconnected from the power supply. When the isotachophoretic zones enter the analytical capillary channel, the mode switches to zone electrophoresis and an online detector detects analytes for qualitative and quantitative analysis.
Claims
1. A device for separation and analysis of ionogenic compounds comprising a separation chip, a multichannel power-supply, a detection system, and a system for data acquisition and analysis, where said separation chip comprises a. a separation channel, b. a tapered channel, c. at least two side channels connected to said tapered channel, d. a restoration capillary channel, e. a side isotachophoretic capillary channel, f. an analytical capillary channel, g. a terminating electrolyte reservoir, h. side leading electrolyte reservoirs connected to said side channels, i. a leading electrolyte reservoir connected to said side isotachophoretic capillary channel, and j. at least one background electrolyte reservoir, where said separation channel is connected to said terminating electrolyte reservoir on a first side and to said tapered channel on a second side that is opposite the first side: said tapered channel is connected on its sides to said side channels while said side channels are connected to said side leading electrolyte reservoirs and said tapered channel is connected at its narrow end to said restoration capillary channel; said restoration capillary channel is connected to said analytical capillary channel, said analytical capillary channel is further connected to said background electrolyte reservoir, said analytical capillary channel is also connected via said side isotachophoretic capillary channel to said leading electrolyte reservoir.
2. A device for separation and analysis of ionogenic compounds according to claim 1, where said separation chip is made from an electrically insulated material selected from group consisting of borofloat glass, fused silica, silicon, poly(methyl methacrylate), polycarbonate, and cyclic polyolefins.
3. A device for separation and analysis of ionogenic compounds according to claim 1 further comprising an injection device.
4. A device for separation and analysis of ionogenic compounds according to claim 1, where the width of said separation channel is at least forty times larger than the width of said analytical capillary channel.
5. A device for separation and analysis of ionogenic compounds according to claim 1, where said side channels and said tapered channel are interconnected through constrictions of said side channels.
6. A device for separation and analysis of ionogenic compounds according to claim 1, where said separation channel, said tapered channel, said side channels, said restoration capillary channel, and said analytical capillary channel have a permanent wall coating.
7. A device for separation and analysis of ionogenic compounds according to claim 1, where at least three pairs of said side channels are connected to said tapered channel; the first pair of said side channels is connected to the end of said separation channel and the beginning of said tapered channel.
8. A device for separation and analysis of ionogenic compounds according to claim 1, where sensing electrodes of a tell-tale detector are connected to said separation channel.
9. A device for separation and analysis of ionogenic compounds according to claim 1, where sensing electrodes of a tell-tale detector are connected to said tapered channel.
10. A device for separation and analysis of ionogenic compounds according to claim 1, where the height of said separation channel, said tapered channel, and said analytical capillary channel ranges from about 30 m to about 100 m, the width of said separation channel ranges from about 10 mm to about 40 mm, and the width of said analytical capillary channel ranges from about 60 m to about 200 m.
11. A device for separation and analysis of ionogenic compounds according to claim 1, where sensing electrodes of a tell-tale detector are connected to said restoration capillary channel.
12. A method to separate and analyze ionogenic compounds in a device according to claim 1, where: a. prior to the analysis, said separation channel, said tapered channel, said side channels, said restoration capillary channel, said side isotachophoretic capillary channel, and said leading electrolyte reservoirs are filled with a leading electrolyte, said terminating electrolyte reservoir is filled with a terminating electrolyte, and said background electrolyte reservoirs and said analytical capillary channel are filled with a liquid; b. a sample is loaded; c. voltage is applied to electrodes in said leading electrolyte reservoirs and said terminating electrolyte reservoir to perform isotachophoresis of ionogenic analytes in said separation channel; d. the presence of isotachophoretic zones of said analytes is detected by electrodes of a tell-tale detector; e. when a rare boundary of said leading ion is detected at the end of said separation channel, the voltage applied to electrodes in a first pair of said side leading electrolyte reservoirs is changed to provide electric current of reversed polarity for the time necessary to let said isotachophoretic zones of said analytes migrate just behind a mouth of a first pair of side channels then a voltage is applied to the electrodes in said first pair of side leading electrolyte reservoirs to turn off said electric current of reversed polarity towards said electrodes in said first pair of side leading electrolyte reservoirs; f. isotachophoretic zones of said analytes migrate further into said tapered channel and the voltage applied to electrodes in a next second pair of side leading electrolyte reservoirs is changed to provide electric current of reversed polarity for the time necessary to let said isotachophoretic zones of said analytes migrate just behind a mouth of a second pair of side channels, then a voltage is applied to said electrodes in said second pair of side leading electrolyte reservoirs to turn off said electric current of reversed polarity towards said electrodes in said second pair of side leading electrolyte reservoirs; g. when said rear boundary of said leading ion zone is detected by said tell-tale detector at the end of said tapered channel, the voltage applied to electrodes in a last pair of side leading electrolyte reservoirs is changed to provide electric current of reversed polarity for the time necessary to let said isotachophoretic zones of said analytes migrate just behind a mouth of a last pair of side channels, then the voltage is applied to said electrodes in said last pair of side leading electrolyte reservoirs to turn off said electric current of reversed polarity towards said electrodes in said last pair of side leading electrolyte reservoirs; h. the isotachophoretic zones of said analytes then enter said restoration capillary channel and isotachophoretic boundaries between them, the sharpness of which was compromised by previous migration through said tapered channel, are restored by isotachophoretic migration in said restoration channel; i. isotachophoretic zones of said analytes are further moved into said analytical capillary channel and further processed.
13. A method to separate and analyze ionogenic compounds according to claim 12, where said ionogenic compounds are separated and concentrated by isotachophoretic migration in said separation channel at constant current.
14. A device for separation and analysis of ionogenic compounds according to claim 1, also comprising a side zone-electrophoresis capillary channel that is connected to said restoration capillary channel and to a background electrolyte reservoir.
15. A device for separation and analysis of ionogenic compounds according to claim 3, where said injection device is a 4-way injection valve.
16. A method to separate and analyze ionogenic compounds according to claim 12, where a modified voltage was applied for 0 to 20 s to provide said electric current of reversed polarity.
17. A method to separate and analyze ionogenic compounds according to claim 12, where said modified voltage to provide said electric current of reversed polarity is set at the value that generates said electric current of reversed polarity at 0-20% of the absolute value of the original driving current.
18. A method to separate and analyze ionogenic compounds according to claim 12, where, after turning off said electric current of reversed polarity, said modified voltage is applied to provide electric current of original polarity at 0-20% of the original value.
19. A method to separate and analyze ionogenic compounds according to claim 12, where said liquid is a background electrolyte and isotachophoretic zones of said analytes migrate through said analytical capillary channel in zone-electrophoresis mode and said analytes are detected by an analytical detector.
20. A method to separate and analyze ionogenic compounds according to claim 12, where said liquid is a leading electrolyte and isotachophoretic zones of said analytes migrate through said analytical capillary channel in isotachophoretic mode and said analytes are detected by an analytical detector.
21. A device for separation and analysis of ionogenic compounds according to claim 1, where sensing electrodes of an analytical detector are connected to said analytical capillary channel.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
DETAILED DESCRIPTION OF THE INVENTION
(4) We disclose a device for separation and analysis of ionogenic compounds comprising a separation chip, a multichannel power-supply, a detection system, and a system for data acquisition and analysis, where said separation chip comprises
(5) a. a separation channel,
(6) b. a tapered channel,
(7) c. at least two side channels connected to said tapered channel,
(8) d. a restoration capillary channel,
(9) e. a side isotachophoretic capillary channel,
(10) f. an analytical capillary channel,
(11) g. a terminating electrolyte reservoir,
(12) h. side leading electrolyte reservoirs connected to said side channels,
(13) i. a leading electrolyte reservoir connected to said side isotachophoretic capillary channel, and
(14) j. at least one background electrolyte reservoir,
(15) in the way that said separation channel is connected to said terminating electrolyte reservoir on one side and to said tapered channel on the other side; said tapered channel is connected on its sides to said side channels while said side channels are connected to said side leading electrolyte reservoirs and said tapered channel is connected at its narrow end to said restoration capillary channel; said restoration capillary channel is connected to said analytical capillary channel, when said analytical capillary channel is further connected to said background electrolyte reservoirs, when said analytical capillary channel is also connected via said side isotachophoretic capillary channel to said leading electrolyte reservoir connected to said side isotachophoretic capillary channel.
(16) A device is also disclosed to separate and analyze ionogenic compounds where said separation chip is made from an electrically insulated material selected from group consisting of borofloat glass, fused silica, silicon, poly(methyl methacrylate), polycarbonate, and cyclic polyolefins.
(17) Further we disclose device for separation and analysis of ionogenic compounds, where that comprises an injection device.
(18) We also disclose a device for separation and analysis of ionogenic compounds, where the width of said separation channel is at least forty times larger than the width of said analytical capillary channel.
(19) A device for separation and analysis of ionogenic compounds is also disclosed where said side channels and said tapered channel are interconnected through constrictions of said side channels.
(20) A device for separation and analysis of ionogenic compounds is also disclosed, where said separation channel, said tapered channel, said side channels, said restoration capillary channel, and said analytical capillary channel have a permanent wall coating.
(21) Further we disclose a device for separation and analysis of ionogenic compounds, where at least three pairs of said side channels are connected to said tapered channel when the first pair of said side channels is connected to the end of the separation channel and the beginning of said tapered channel.
(22) We also disclose a device for separation and analysis of ionogenic compounds, where sensing electrodes of a tell-tale detector are connected to said separation channel.
(23) A device for separation and analysis of ionogenic compounds is also disclosed, where sensing electrodes of a tell-tale detector are connected to said tapered channel.
(24) Further we disclose a device for separation and analysis of ionogenic compounds, where the height of said separation channel, said tapered channel, and said analytical capillary channel ranges from about 30 m to about 100 m, the width of said separation channel ranges from about 10 mm to about 40 mm. and the width of said analytical capillary channel ranges from about 60 m to about 200 m.
(25) We also disclose a device for separation and analysis of ionogenic compounds, where sensing electrodes of a tell-tale detector are connected to said restoration capillary channel.
(26) A method to separate and analyze ionogenic compounds in a microfluidic device is also disclosed, where a. prior the analysis, said separation channel, said tapered channel, said side channels, said restoration capillary channel, said side isotachophoretic capillary channel, and said leading electrolyte reservoirs are filled with said leading electrolyte, said terminating electrolyte reservoir is filled with said terminating electrolyte, and said background electrolyte reservoirs and said analytical capillary channel are filled with a liquid; b. a sample is loaded; c. voltage is applied to electrodes in said leading electrolyte reservoirs and said terminating electrolyte reservoir to perform isotachophoresis of ionogenic analytes in said separation channel; d. the presence of isotachophoretic zones of said analytes is detected by said electrodes of said tell-tale detectors; e. when the rare boundary of said leading ion is detected at the end of said separation channel, the voltage applied to the electrodes in the first pair of said side leading electrolyte reservoirs is changed to provide electric current of reversed polarity for the time necessary to let said isotachophoretic zones of said analytes migrate just behind the mouth of said side channels, then a voltage is applied to said electrodes in said first pair of said side leading electrolyte reservoirs to turn off said electric current of reversed polarity towards said electrodes in said first pair of said side leading electrolyte reservoirs; f. isotachophoretic zones of said analytes migrate further into said tapered channel and the voltage applied to the electrodes in the next pair of said leading electrolyte reservoirs is changed to provide electric current of reversed polarity for the time necessary to let said isotachophoretic zones of said analytes migrate just behind the mouth of said side channels, then a voltage is applied to said electrodes in said pair of said side leading electrolyte reservoirs to turn off said electric current of reversed polarity towards said electrodes in said pair of said side leading electrolyte reservoirs; g. when said rear boundary of said leading ion zone is detected by said tell-tale detector at the end of said tapered channel, the voltage is applied to the electrodes in the last pair of said side leading electrolyte reservoirs to provide electric current of reversed polarity for the time necessary to let said isotachophoretic zones of said analytes migrate just behind the mouth of said side channels, then the voltage is applied to said electrodes in said pair of said side leading electrolyte reservoirs to turn off said electric current of reversed polarity towards said electrodes in said pair of said side leading electrolyte reservoirs; h. the isotachophoretic zones of said analytes then enter said restoration capillary channel and isotachophoretic boundaries between them, the sharpness of which might have been compromised by previous migration through said tapered channel, are restored by isotachophoretic migration in said restoration channel; i. isotachophoretic zones of said analytes are further moved into said analytical capillary channel and further processed.
(27) Further we disclose a method to separate and analyze ionogenic compounds, where said ionogenic compounds are separated and concentrated by isotachophoretic migration in said separation channel at constant current.
(28) We also disclose a device for separation and analysis of ionogenic compounds according to claim 1, also comprising a side zone-electrophoresis capillary channel that is connected to said restoration capillary channel and to a background electrolyte reservoir.
(29) A device for separation and analysis of ionogenic compounds according to claim 3 is also disclosed, where said injection device is a 4-way injection valve.
(30) We disclose a method to separate and analyze ionogenic compounds according to claim 12, where said voltage applied to said electrodes to provide said electric current of reversed polarity is applied for 0-20 s.
(31) A method to separate and analyze ionogenic compounds according to claim 12 is disclosed, where said voltage applied to said electrodes to provide said electric current of reversed polarity is set at the value that generates said electric current of reversed polarity at 0-20% of the absolute value of the original driving current.
(32) We further disclose a method to separate and analyze ionogenic compounds according to claim 12, where, after turning off said electric current of reversed polarity, said voltage is applied to said electrodes to provide electric current of original polarity at 0-20% of the original value.
(33) A method to separate and analyze ionogenic compounds according to claim 12 is also disclosed, where said liquid is said background electrolyte and isotachophoretic zones of said analytes migrate through said analytical capillary channel in zone-electrophoresis mode and said analytes are detected by said analytical detector.
(34) We also disclose a method to separate and analyze ionogenic compounds according to claim 12, where said liquid is said leading electrolyte and isotachophoretic zones of said analyte migrate through said analytical capillary channel in isotachophoretic mode and said analytes are detected by said analytical detector.
(35) We further disclose a device for separation and analysis of ionogenic compounds according to claim 1, where sensing electrodes of an analytical detector are connected to said analytical capillary channel.
EXAMPLES
Example 1
Preparation of Glass Chips
(36) The chip was prepared from borofloat glass wafers 1.1 mm thick with diameter of 10.0 mm. After cleaning the wafers with piranha solution (mixture of 96% sulfuric acid and 30% hydrogen peroxide 9:1) at 120 C. for 20 min, a layer of amorphous silicon was applied in a furnace at 525 C. for 240 min in the presence of SiH.sub.4. Then the wafers were coated with hexamethyl disilazane at 110 C. and Shipley photoresist 3612 was applied. A Mylard mask with all the structures drawn on it was used to expose the photoresist layer with UV lamp for 10 s. The exposed photoresist layer was developed and the exposed silicon layer was removed by dry plasma etching in the presence of SF.sub.6 and chlorodifluoromethane (F22). Channels were etched with 49% HF at room temperature for 4.5-9 min. Holes of diameter 1.4 mm and 2.0 mm connecting the channels with outer world were drilled with diamond drills on wafers not coated with amorphous silicon. After cleaning all the wafers with piranha solution (mixture of 96% sulfuric acid and 30% hydrogen peroxide 9:1) at 120 C. for 20 min, the upper wafers with drilled holes and lower wafers with etched channels were bonded by anode bonding at 240-300 C. and 1,200 V for 30 min.
(37) Platinum wire with a diameter of 25-75 m was rolled over with a steel cylinder to make their cross-section elliptic. Then they were inserted into corresponding channels to serve as electrodes for conductivity detection. The sensing electrodes were fixed there by filling the channels with a UV-curable epoxy glue Ultra Light-Weld 3069 (Dymax) and illumination with a UV lamp for 180 s. The electrode reservoirs were attached with the same UV curable epoxy glue.
Example 2
Chip for Isotachophoresis-Zone Electrophoresis
(38) The chip for isotachophoresis-zone electrophoresis on chip contained said separation channel, which was 16 mm wide and 48 mm long. The height of said separation channel as well as the height of all other channels was approx. 45 m. There was a sampling four-way valve V placed at the beginning of the separation channel, filled with the sample from said sampling inlet S towards said waste outlet W, behind said terminating electrolyte reservoir. In some setups, said sampling valve was eliminated and high-density samples were simply injected at the bottom of said terminating electrolyte. At the end of said separation channel, a pair of sensing electrodes for said tell-tale detector made from 25-75 m platinum wire were attached. Behind said separation channel, said tapered channel followed, 24 mm long, reducing the cross section from 16 mm to approx. 130 m. Four pairs of said side channels connected said tapered channel and leading electrolyte reservoirs. The dimensions of said side channels (length 10-25 mm, width 1 mm) were selected not to significantly contribute to resistance of the electric circuit and simultaneously suppress hydrodynamic flow between said electrolyte reservoirs. Constrictions of said side channels at the connection to said tapered channel suppressed loss of analytes by diffusion out of said tapered channel. Said side channels were connected to said tapered channel with an uneven distribution with a shorter distance between them at the narrower part of said tapered channel. At the end of said tapered channel, said restoring channel (130 m wide, approx. 40 mm long) was connected. Sharp isotachophoretic boundaries that might have been disturbed during their migration through said tapered channel were restored here. Said restoring channel was connected on the other end to said analytical channel (130 m wide, effective length approx. 150 mm). A pair of sensing electrodes for said tell-tale detector was attached in front of said leading electrolyte reservoir E, i. e., at the end of said restoring channel or at the beginning of said analytical channel. Three electrolyte reservoirs were connected to said analytical channel: said leading electrolyte reservoir via side isotachophoretic capillary channel, said background electrolyte reservoir F via side zone-electrophoretic capillary channel, and said background electrolyte reservoir G. At the end of said analytical capillary channel, a pair of sensing electrodes made from 25-75 m platinum wire was connected as said analytical detector. A C4D detector was also used in some setups as said analytical detector. In some experiments, said channels were coated with a galactomannan coating (U.S. Pat. No. 7,799,195) to eliminate electroosmotic flow during analysis.
Example 3
Electrolytes Used in the Analysis
(39) Leading Electrolytes LE 1) 50 mM HCl, 100 mMalanine, 4 g/L hydroxyethyl cellulose (HEC) 2) 50 mM HCl, 100 mMalanine, 4 g/L hydroxyethyl cellulose (HEC), 50 g/Lcyclodextrin 3) 50 mM HCl, 100 mMalanine, 50 g/L dextran, M.sub.w210.sup.6 4) 50 mM HCl, 150 mMalanine, 4 g/L hydroxyethyl cellulose (HEC) 5) 50 mM HCl, 150 mMalanine, 4 g/L hydroxyethyl cellulose (HEC), 50 g/Lcyclodextrin 6) 50 mM HCl, 50 mMalanine, 50 g/L dextran, M.sub.w210.sup.6
(40) Terminating Electrolyte TE 1) 100 mM glycolic acid 2) 100 mM NaH.sub.2PO.sub.4 3) 100 mM mandelic acid 4) 20 mM mandelic acid
(41) Background Electrolytes BGE 1) 10 mM HCl, 20 mMalanine, 40 g/L -cyclodextrin 2) 10 mM HCl, 10 mMalanine, 40 g/L -cyclodextrin 3) 10 mM HCl, 20 mMalanine, 100 g/L poly(vinyl pyrrolidone), M.sub.w40,000 4) 10 mM HCl, 10 mMalanine, 100 g/L poly(vinyl pyrrolidone), M.sub.w40,000, 5) 10 mM HCl, 20 mMalanine, 100 g/L poly(vinyl pyrrolidone), M.sub.w40,000, 40 g/L -cyclodextrin 6) 10 mM HCl, 10 mMalanine, 100 g/L poly(vinyl pyrrolidone), M.sub.w40,000, 40 g/L -cyclodextrin
Example 4
(42) Voltage and Current Applied to HVS4448 During Steps 1-Step 6
(43) The analysis by isotachophoresis-zone electrophoresis was typically performed in 6 independent steps. The values for constant current and constant voltage are summarized in Table 1. To speed equilibration of current, initial approximate voltage values were applied at the beginning of a step followed by target constant current values.
(44) TABLE-US-00001 TABLE 1 Initial voltage and current values applied Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Electrode A 0 V 0 A 0 A 0 A 0 A 0 A Electrode B 600 A 600 A 0 A 0 A 0 A 0 A Electrode C 400 A 400 A 400 A 0 A 0 A 0 A Electrode D 360 A 360 A 360 A 360 A 0 A 0 A Electrode E 60 A 0 V 0 V 0 V 0 V 0 A Electrode F 0 A 0 A 0 A 0 A 0 A 3,000 V Electrode G 0 A 0 A 0 A 0 A 0 A 3,000 V Electrode H 2220 A 1500 A 900 A 420 A 80 A 0 A
Example 5
(45) Performing Isotachophoresis-Zone Electrophoresis on Chip Analysis
(46) First, said analytical capillary channel and said background electrolyte reservoirs were filled with background electrolyte, then said tapered channel, all said side channels, all said leading electrolyte reservoirs, and said separation channel were filled with leading electrolyte. Then said terminating electrolyte reservoir was filled with terminating electrolyte and a sample was applied either with the four-way valve or simply by pipetting it on the bottom of said terminating electrolyte reservoir. When electrodes were connected, a sequence of steps with an 8-channel power supply HVS448 (LabSmith, Inc., Livermore, Calif.) was started. In Step 1, ITP separation was performed. Said tell-tale detector (a contact conductivity detector, Villa Labeco, Spissk Nov Ves, Slovakia) indicated the end of Step 1. Using a comparator LM393P (Texas Instrument, Dallas, Tex.) , a TTL/LTT impuls was sent to the HVS448 and voltages and currents were switched as programmed for Step 2. In Step 2, migration towards electrodes A was disconnected and a low current of 40 A or less was applied to prevent analytes from entering said side channels connected to said electrodes A. Duration of Step 2, i.e., the time necessary for the colored ITP zone to get in front of said side channels connected to said electrodes B was predetermined with a high-mobility colored marker 2,7-naphthalenedisulfonicacid,4,5-dihydroxy-3-((4-sulfophenyl)azo)-,trisodium (SPADNS). After this time, the sequence switched to Step 3. In Step 3, migration towards electrodes B was stopped and a low current of 40 A or less was applied on said electrodes B to prevent analytes from entering said side channels connected to said electrodes B. Duration of Step 3 was also predetermined from migration of colored zone of SPADNS. At the end of Step 3, said sequence moved to Step 4 with voltage and current applied as listed in Table 1. Said tell-tale detector (a contact conductivity detector, Villa Labeco, Spissk Nov Ves, Slovakia) indicated the end of Step 4. Using a comparator LM393P (Texas Instrument, Dallas, Tex.), a TTL/LTT impuls was sent to the HVS448 and voltages and currents were switched to progress to Step 5. Said tell-tale detector (a contact conductivity detector, Villa Labeco, Spissk Nov Ves, Slovakia) indicated then the end of Step 5. Using a comparator LM393P (Texas Instrument, Dallas, Tex.), a TTL/LTT impuls was sent to the HVS448 and voltages and currents were switched and Step 6 started. During Step 6, analytes migrated in the zone electrophoretic mode towards the destination electrode in said baground electrolyte reservoir G and their zones were detected by analytical detector, either contact conductivity detector (Villa Labeco, Spissk Nov Ves, Slovakia) or C4D detector (J. A. Fracassi da Silva and C. L. do Lago: An Oscillometric Detector for Capillary Electrophoresis. Anal. Chem., 1998, 70, 4339-4343) with wave generator based on the headstage ET121 and C4D detector ER125 (eDAQ, Ltd., Australia).
Example 6
Analysis of Perchlorate in Body Fluids
(47) Samples were ultrafiltered through Amicon Ultra centrifugal filters (0.5 mL, 30 k, Millipore) by centrifugation at 14,000 RPM for 30 min. Said capillary channels and said corresponding electrolyte reservoirs (E, F, G) were filled with BGE (10 mM HCl, 10 mMalanine, 100 mM poly(vinyl pyrrolidone), M.sub.w 40,000), said separation channel, said tapered channel, said side channels and said corresponding leading electrolyte reservoirs (A, B, C, and D) were filled with LE (50 mM HCl, 100 mMalanine, 50 g/L dextran, Mw 2,000,000), and said terminating electrolyte reservoir (H) with terminating electrolyte (20 mM mandelic acid in 18 M water). 10 L samples were pipetted on the bottom of said terminating electrolyte reservoir and the analysis was performed according to Example 5.