Introduction to Capillary Electrophoresis Technology

1. Introduction to Capillary Electrophoresis Technology

2. Outline • Overview of Capillary Electrophoresis (CE) • Modes of CE operation • Development History – Parallel CE with UV Absorbance Detection • cePRO 9600™ Technology • Benefits of Parallel CE Technology • Summary

3. Inlet (injection) Side Outlet Side Introduction to Capillary Electrophoresis (CE) • Separation by applying high voltage across fused silica capillary filled with conductive buffer • Capillary can be filled with: • Aqueous buffer (CZE) • Aqueous-based buffer with surfactants (MEKC, MEEKC) • Gel matrix (CGE) • Detection can be by UV absorbance, fluorescence, mass spectrometry, conductivity

4. Electro osmotic Flow (EOF) • In a bare, uncoated silica capillary, a double layer of ions forms at pH > 4 • The outer mobile layer is driven to cathode (detector), creating bulk flow • This bulk flow can carry both positive and negative analytes to detector • EOF must be eliminated for some applications such as DNA/protein separations

5. -- - - + ++ + N - -- + ++ Bulk Flow: EOF + Vacuum Time UV Capillary Zone Electrophoresis (CZE) • Application of high voltage across capillary filled with aqueous conductive buffer • Narrow bore, fused silica capillaries (50-75 mm i.d., 192 mm o.d.) • Electroosmotic flow (EOF) provides bulk flow towards cathode (detector) at pH > 4 • Application of vacuum can provide additional bulk flow to detector at all pH values • Migration time dependent on analyte’s charge-to-mass ratio; neutral compounds migrate with bulk flow

6. Least Hydrophobic Most Hydrophobic EOF + - Time Micellar Electrokinetic Chromatography (MEKC) • Separation based on the partition of analytes between the aqueous phase and charged micelle phase with ionic headgroup and hydrophobic core (e.g., SDS) • More hydrophilic/lipophilic compounds favor the micelle phase and migrate slower • A retention window is created with order of migration: EOF marker (e.g., DMSO), analyte, micelle marker (e.g., dodecylbenzene) • Separation of neutral/charged compounds based on hydrophobicity/charge state • MEKC is the “CE version” of reversed-phase HPLC Figure taken from presentation at http://franklin.chm.colostate.edu/cshenry/C531.ppt

7. - - - - - - + - - - - UV Capillary Gel Electrophoresis (CGE) • Capillary is filled with conductive gel sieving matrix • Size-based separation of species possessing a constant mass-to-charge ratio (e.g., denatured SDS-protein complexes, ssDNA oligonucleotides, dsDNA/dsRNA) • Smaller sized species migrate faster, larger molecules move slower • EOF needs to be eliminated to achieve maximum separation performance • CGE is “CE version” of slab gel electrophoresis (PAGE)

8. Vacuum (-0.2 psi) Sample Plug Vacuum Injection of Samples in CE • Advantages: • No injection bias of sample molecules due to sample charge • Sample matrix does not influence amount of sample injected • Disadvantages: • Little to no sample “stacking” effects = poorer sensitivity • More difficult to reproducibly control • Difficult to inject samples into high viscosity buffers (e.g., gel matrix) Vacuum injection is suitable for most CE applications: pKa, log P, chiral separations, protein sizing

9. Capillary with high conductivity buffer Capillary with high conductivity buffer + + Cl ˉ Cl ˉ Cl ˉ Cl ˉ Cl ˉ - - - - - - - - - - Electrokinetic (Voltage) Injection of Samples in CE Interface Low conductivity sample (e.g. H2O) High conductivity sample (e.g. Tris-HCl) • Advantages: • “Stacking”: sample ions focus at low conductivity sample/high conductivity buffer interface • Stacking of sample ions can greatly increase detection sensitivity • Disadvantages: • Injection bias: only ions possessing appropriate charge will be injected • Matrix effects: sample will not be injected in presence of salts Electrokinetic injection is used for DNA analysis

10. Multiplexed (Parallel) CE-UV Technology • 24 or 96 capillaries are arranged in a linear array at detection window • UV light is passed through capillary array and imaged onto photodiode array detector • Capillary inlets are arranged 8 x 12 for direct sample injection from 96-well micro plates • Capillary outlets are bundled to a common reservoir connected to pumping system • Sample injection by vacuum or voltage • 24 or 96 individual CE separations are performed in parallel

11. W lamp (idle) camera lens fan window diode array Hg lamp light shield battery samples flush/ fill Dr. Edward Yeung’s Laboratory Prototype (“Gray Box”) • Manual capillary filling (by syringe); manual sample injection (by gravity or voltage) • Proof-of-concept demonstrated (Gong and Yeung, Anal. Chem.1999, 71, 4989-4996) • Patent protected, core technology exclusively licensed from ISU to CombiSep

12. MCE 2000System • First generation commercial 96-capillary array CE-UV instrument • Fixed wavelength UV or visible detection; vertical optics design • Vertical z-stage accommodates a single 96-well plate • User manually exchanges waste, buffer and sample plates during analysis

13. cePRO 9600™ System • Second generation 96-capillary array CE instrument • Horizontal optics design; slightly improved detection sensitivity • Automated XYZ stage holds up to four 96-well plates (1 waste, 1 buffer, 2 sample) • System can be interfaced to a robotic arm for unattended well plate exchange

14. HV Power Supply Capillary Array Detection Window Capillary Array Cartridge Optical Platform Housing Lamp Housing Syringe Pump Inside View of the cePRO 9600™ Instrument

15. 96-Capillary Array Viewed from Detector Position Capillary Outlets (12 Bundles of 8 Capillaries) Capillary Inlets (Arranged in 8 x 12 Format) Detection Window (Polyimide coating removed)

16. Capillary Array Inlets Viewed from Below • Capillaries are arranged side-by-side with tungsten electrodes • Direct injection by vacuum or voltage from 96-well plates • Recommended minimum sample volume: 40 mL

17. Image of 96-Capillary Array on PDA Detector • Continuous measurement of UV intensity simultaneously in all 96 capillaries • Absolute light intensity does not have to be equal as the relative absorbance is measured in each capillary

18. cePRO 9600 Performance Specifications • Detection • Fixed wavelength, using D2 (193 nm – 400 nm), Zn (214 nm) Cd (228 nm) or Hg (254 nm) lamps and narrow band pass filter • Capillary Array Dimensions • 50 mm or 75 mm i.d., 200 mm o.d. • Effective lengths 30 cm to 60 cm; 20 cm fixed length from detector to outlet • Sample Injection • Vacuum or Electrokinetic; 40 mL minimum sample volume; nL volumes injected • Operational Conditions • Typical total operating current < 4 mA (<40 mA/capillary) • Operating voltage to ± 16 kV; field strength to 250 V/cm • Forced air cooling at RT or at ~16 ºC • Detection Limits • Low mM concentrations for a standard injection volume • Common MCE-UV Separation Modes • Capillary Zone Electrophoresis (CZE) • Microemulsion Electrokinetic Chromatography (MEEKC) • Capillary Gel Electrophoresis (CGE)

19. Robotic Arm Integration for Unattended Analysis • A Thermo CRS Catalyst Express robotic arm was integrated to facilitate unattended well plate exchange between multiplexed CE-UV runs. • Using this configuration, 96 samples were analyzed in parallel over 24 pH values in 8 h (2304 total separations) to determine their respective pKa values without user intervention. Only 10 mg of compound was required for the analysis. • Use of a robotic arm interfaced with MCE-UV and a well plate hotel allows for long term and/or overnight operation.

20. Benefits of Multiplexed CE-UV for Pharmaceutical and Biotechnology Applications • High Throughput - simultaneous monitoring of up to 96 individual CE separations • UV absorbance (214 nm, 254 nm) provides more universal analyte detection • Variation of buffer conditions (pH, ionic strength, additives) in different capillaries for accelerating methods development • Multiple applications can be performed using the same instrument platform • Minimal sample consumption (g amounts required; ng amounts consumed) • Sample impurities can be resolved when performing assays • Low cost per sample

21. Application Specific System Solutions Oligo PRO™ System pKa PRO™ System • Parallel CGE system • ssDNA/ssRNA purity analysis • dsRNA duplex analysis • High pressure pumping system (500 psi) • Customized, application specific software • 24 or 96 capillary configurations • Parallel CZE/MEKC system • pKa and pI measurements • Dedicated software for pKa data analysis • Also for log P analysis; chiral separations • 24 or 96 capillary configurations

22. Summary • Multiplexed CE-UV technology can be applied to a broad range of high throughput applications spanning pharmaceutical and biotechnology markets • The ability to perform CE in parallel with 24 or 96 capillary arrays provides a significant improvement in throughput and laboratory efficiency at a fraction of the cost for alternative technologies • Many methods previously developed for single capillary CE instruments can be successfully transferred to a multiplexed format • The multiplexed CE format provides the flexibility to simultaneously vary separation conditions to speed development processes • Multiplexed CE-UV provides a significant business advantage: low cost per sample, lower turnaround times, small sample consumption, decreased reagent consumption, and increased throughput