Basics of Capillary Electrophoresis Ronald E. Majors Hewlett-Packard Wilmington, DE
Outline Characteristics of CE EOF Phenomena Modes of Operation Zone Broadening Mechanisms Detection Modes
Characteristics of CE (I) Electrophoresis is performed in narrow-bore (25 150 µm id), fused silica capillaries Operates in aqueous media Small sample volume required (1 to 50 nl injected) High voltages (10 to 30 kv) and high electric fields (100 to 1000 V/cm) are applied across the capillary High resistance of the capillary limits current generation and internal heating
Characteristics of CE (II) High efficiency (N>10E5 to 10E6 ) and short analysis time MEKC CGE CZE IEF ITP Numerous modes to vary selectivity and wide application range Simple method development Automated instrumentation Detection performed in 2 modes: on-capillary and decoupled detection cell
Principle of CE Detector Inlet reservoir Exit reservoir Total length Effective length
Methods of Sample Injection Pressure Hydrodynamic Electrokinetic Sample Sample Vacuum Siphoning Sample Sample
Instrumental Aspects of CE Capillary thermostatting Diode-array detector Capillary cartridge HV Schematic of CE instrumentation Vial carousel (thermostatted) Buffer replenishment
Application Areas of CE Pharmaceutical low MW molecules, charged and neutrals, chirals reaction intermediates, purity validation, stability, final product testing Bioscience peptides, proteins, DNA, carbohydrates Foods inorganic cations/anions, organic acids, amino acids, carbohydrates Environmental/Chemical pesticides, PNAs, inorganic ions, transition metals, surfactants, dyes
Development of the Electroosmotic Flow (EOF) Negatively charged fused silica surface (Si-O ) Hydrated cations accumulate near the surface Bulk flow is towards the cathode upon application of the electric field
Effect of Flow Profile on Zone Width EOF Laminar flow
µ EOF 4 3 Effect of ph on EOF in Various Capillary Materials 10 cm [ - 4 2 V s ] Pyrex Silica 2 1 Teflon 3 4 5 6 7 8 ph
Modes of Operation (I) Mode Capillary zone electrophoresis (CZE) Micellar electrokinetic chromatography (MEKC) Capillary gel electrophoresis (CGE) Basis of separation Free solution / mass to charge ratio (FS/coated capillaries) Hydrophobic/ionic interactions with micelles / retardation Size and charge
Modes of Operation (II) Mode Basis of separation Isoelectric focusing(ief) Isotachophoresis (ITP) Capillary Electrochromatogaphy (CEC) Isoelectric point Moving boundaries interaction with solid phases / retardation
Mechanism of Separation for Zonal Electrophoresis Zone electrophoresis t = 0 t > 0 performed in bare fused silica capillaries and in permanently or dynamically coated capillaries
Capillary Zone Electrophoresis (CZE): Migration Superimposed on EOF in Fused Silica EOF
Mobility Calculation of EOF and Solute mau (93.1 s) Solute Migration time [s] µ a[cm /Vs] µ e[cm /Vs] 2 2 Cation 38.4 3.05 10-3 7.40 10 Neutral 50.7 2.31 10 2.31 10-3 Anion 93.1 1.26 10-1.05 10-3 -4-3 -3 (38.4 s) (50.7 s) Cation: Neutral: µ a ll Vt 50 58. 5 = = = 25000 38. 4 50 58. 5 µ EOF = =. 25000 50. 7 3. 05 10 3 2 31 10 3 0.5 1.0 1.5 2.0 µ = µ µ =.. =. e a EOF 3 05 10 2 31 10 7 40 10 3 3 4 (Note: µ will be negative for the anion) e v= ion velocity; u = electoosmotic velocity (cm /V 2 ) ; E= applied electric field (V/cm);
Capillary Zone Electrophoresis (CZE) in neutral coated capillary
CZE-Separations in PVA Capillaries ph3 mau ph 9 12 15 10 5 Procainamide Tocainide Diisopyramide 10 8 6 4 2 Pepsin inhomogen Pepsin Trypsin Inhibitor α-lactalbumin Myoglobin Carbonic Anhydrase II 0 0 2 4 6 8 10 12 min 10 20 30 40 capillary: PVA, Leff = 56 cm, Ltot = 64.5 cm, i.d. 50 µm; run buffer = 50 mm phosphate ph 3/ 50 mm 2-amino-2- methyl-1,3-propandiol ph 9(AMPD) ph 9; flush = 2 min run buffer; injection: sample =125 mbars, run buffer (postinjection) = 200 mbars, separation: voltage = +/-30 kv, temperature = 20 º C, detection: 215 (8) nm min
Micellar Electrokinetic Chromatography (MEKC) EOF
Elution Time Window for Neutral Solutes in MEKC Time window Solutes EOF Micelle 0 t t R t t t 0 1 R2 R 3 m Time
Volts 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0 MEKC of a Forensic Drug Screen b c d e j a f h g m n r i k q l p o 0 4 8 12 16 20 24 28 32 38 40 44 Psilocybin Morphine Phenobarbital Psilocin Codeine Methaqualone LSD Heroin Amphetamine Librium Cocaine Methamphetamine Lorazepam Diazapam Fentanyl PCP Cannabidiol D-9-THC Borate-phosphate ph 8.5, 85 mm SDS, 15 % acetonitrile (Anal. Chem. 1991, 63, 823
Capillary Gel Electrophoresis (CGE) - Separation based on Size replaceable polymer PVA / CEP coated capillary t < 0 t = 0 t > 0 sieving matrix
mau 40 Oligonucleotide Separation w/out Organic Addititve in Polymer Solution pd(a) 12-18 30 20 without organic additive with organic additive 10 0 5 10 15 20 25 30 35 min separation conditions: capillary: PVA coated, id=100um, leff=24.5 cm; Polymer Solution A/B (w/wo organic additive), neutral Oligonucleotide Buffer; polymer flush: -7.5 bar, 3 min.; sample: oligonucleotide pd(a) 12-18, injection: 7 sec, -10 kv; separation: 30C, voltage =-25 kv; detection: 260 (8) nm, Ref. off, DNA Filter
Separation of DNA Fragments mau 8 2645 bp 1605 bp 6 4 2 36 bp 51 bp 65 bp 75 bp 396 bp 350 bp bp 179 bp222 126 bp 1198 bp 676 bp 460 bp 517 bp 0 8 9 10 11 12 13 14 15 16 17min Capillary: CEP Coated Capillary, l/ L 40/48.5 cm, i.d. 75 µm; Sample: pgem DNA Markers, 1 µg/µl; Buffer: DNA Buffer + 1.5% polymer Injection: -5 kv, 4 s; Voltage: -16.5 kvtemperature: 25 C; Detection: 260 nm with DAD filter for 260 nm (optional)
Isoelectric Focusing (IEF) IEF Coated Capillary filled with mixture of sample and ampholytes t = 0 Low ph ph gradient High ph t > 0
Capillary Isoelectric Focusing (CIEF) ph = 3 ph = 9 pi 3.90 5.01 6.05 7.81
CIEF - Hydrodynamic Mobilisation Comparison of Meat Extracts mau Beef Horse 70 50 30 capillary: PVA, Leff = 56 cm Ltot = 64.5 cm, i.d. = 50 mm sample: meat extracts each 100 ug/ml in glycine injection: whole capillary filled focusing: Servalyt 3-10, 30 kv for 7.5 min, pressure driven mobilization with 50 mbar temp.: 30 C detection:280 nm, reference = off 10 10 11 12 13 14 15 min 10 11 12 13 14 15 16 min
Isotachophoresis ITP T L t = 0 T L t > 0
ITP of Phosphorylated Nucleotides mau 0.3-0.2 10E-7 M Nucleotides in water Injection 5000 mbar s -0.7-1.2 Leading Electrolyte Cl 0.3 5 10 15 20 25 10E-7 M Nucleotides in water + 35 mm NaCl min Trailing Electrolyte MES -0.2-0.7-1.2 5 10 15 20 25 min
Origin of Electro - Osmotic Flow in CEC u eo = σ ε ε 0 r RT 2 2 cf η 1 / 2 E
Capillary ElectroChromatography (CEC): Principles and Practice
Capillary Electrochromatography What is it? CE capillaries filled with HPLC packing Reversed phase or ion exchange material Flow driven by EOF, not an external pump HPLC mobile phases containg a minimal amount of buffer Separation mechanism primarily chromatographic
Benefits of Capillary ElectroChromatography + Separate closely related compounds + CE/MS for neutral species Higher separation efficiency than HPLC and improved selectivity relative to CE Allows separation of neutral compounds without surfactants Minimal sample consumption and low operating costs Offers flexibility to use best separation mode Microscale technique Single instrumentation for both CE and CEC
Principles of Electrochromatography Simplified system for CEC; 1 and 3 buffer vials, 2, FS capillary, 50-100 µm i.d. 250-500 mm length, packed with HPLC packing material, 4 and 6, electrodes, 5, power supply, 7, point of detection.
LC Contributions to Dispersion in a Packed Capillary CEC Pressure drive particle Electroosmotic drive particle velocity profile velocity profile channel channel Radius flow velocity flow velocity
Typical Example of CEC-separation mau 70 60 50 Methylparabene Butylparabene Propylparabene Ethylparabene Pentylparabene Naphthalene Phenanthrene Anthracene Column CEC Hypersil C18, 3 µm 250(350)x0.1 mm Mob. phase 80/20 ACN/MES 25 mm, ph 6 Voltage 25 kv Injection 5 kv, 3 sec Pressure 10 bar both sides Temp. 20 C 40 30 20 10 Thiourea Biphenyl Fluorene Fluoranthene Plate Number 65,000-80,000 Symmetry 0.93-0.98 0 0 1 2 3 4 5 6 7 min
Achievable Efficiencies in µ-hplc and CEC Plates 250,000 200,000 150,000 100,000 50,000 0 µ-hplc 80 70 60 50 40 30 20 10 0 10 20 30 40 50 0 60 Analysis Time [min] Particle size 5 µm 3 µm 1.5 µm Capillary length 50 cm 30 cm 15 cm HPLC N/column 45,000 50,000 33,000 CEC length 50 cm 50 cm 50 cm N/column 90,000 150,000 210,000 Capillary length [cm] 250,000 200,000 150,000 100,000 50,000 0 CEC 80 70 60 50 40 30 20 10 0 10 20 30 40 50 60 0 Analysis Time [min] Conditions: P = 400 bar, f = 1000 in HPLC V = 30 kv, z = 40 mv in CEC t = 25 min for k' = 5 h = 1 cp
CEC Separations on Different Types of RP Packings 2 60 50 40 30 20 10 1 2 1 1 2 3 4 3 3 5 4 4 6 CEC-HYPERSIL Phenyl 5 CEC-HYPERSIL C8 5 CEC-HYPERSIL C18 Columns 3 µm, 250(350)x0.1 mm Mob. phase 80/20 ACN/Tris-HCl 50 mm, ph 8 Voltage 25 kv Injection 5 kv, 3 sec Pressure 10 bar both sides Temp. 20 C k' k' k' C18 MOS PHE 1 Thiourea 2 Dimethylphthalate 0.19 0.19 0.15 3 Diethylphthalate 0.31 0.39 0.20 4 Biphenyl 0.88 0.63 0.33 5 o-terphenyl 1.41 1.03 0.52 6 isomeric esters of Diioctylphthalate 0 2 4 6 8 min
Separation of 14 Explosive Compounds by CEC (Unimicro Technologies) Column: 30 cm X 75-um, packed w/ 1.5-um ODS-II (Micra Scientific) Mobile Phase: 15% MeOH/ 85% 10 mm MES. Appl. Voltage: 12 kv Injection: 1 sec at 2 kv Peaks: 1. HMX 8. Tetryl 2. RDX 9. 2,6-diNO2toluene 3. DNB 10. 2-Am dino2toluene 4. TNB 11. 2-NO2toluene 5. NB 12. 4-NO2toluene 6. TNT 13. 4-AMdiNO2toluene 7. 2,4-dinitrotoluene 14. 3-NO2toluene
Conclusions With CEC, isocratic, reversed phase, µhplc becomes feasible without significant instrumental constraints CEC overcomes high back-pressure difficulties associated with micro- LC with 1.5-3 µm particles With acetonitrile as organic modifier, useful EOF velocities are achieved down to ph 2 The new standard HP3DCE instrument is capable of applying the high pressure necessary to run packed CEC columns under typical conditions (e.g. moderate or high current) The HP3DCE is capable of step-gradient CEC
Mechanisms of Band Broadening in CE Longitudinal diffusion Joule heating Solute-Wall interactions Electrodispersion
Joule Heating and Capillary Thermostatting Current [µa] 300 Ohm's Plot at 25 ºC 50 mm id caillary 250 200 150 Liquid thermostating 10 m/s air thermostating No thermostating 100 50 0 5 10 15 20 25 30 Voltage [kv]
Suppression of Solute - Wall Interactions Suppression dynamic coating - buffer ions - watersoluble polymers - surfactants permanent coating - chemically linked polymers - physically adhered polymers
mau Dymanic Deactivation: Influence of Buffer Concentration on Solute-Wall Interactions 10 mm 50 mm 100 mm capillary: bare fused silica, id = 50 µm l = 50 cm, L = 58.5 cm buffer: phosphate ph 7 voltage: 25 kv current: 9, 36, and 71 µa sample: BSA (2mg/ml) injection: 100 mbars detection: 215 nm 6 8 10 12 14 16 18 20 Time [min]
Separation on permanently coated PVA Capillary mau 2.5 1.5 0.5-0.5 untreated fused silica 0 2 4 6 8 10 12 14 16 18 mau 12 10 8 6 4 2 0 PVA coated capillary min capillary: A - fused silica, B - PVA, both: l = 56 cm, L = 64,5 cm, id = 50mm buffer: 50 mm phosphate ph 3 sample: Cytochrome C, Lysozyme, beta-lactoglobuline A and B injection: 120 mbars sample 200 mbars buffer (post injection) temp.: 20 ºC voltage: 30 kv detection: 215 (8) nm -2 6 8 10 12 14 min
Electrodispersion Due to Mismatched Sample and Buffer Conductivities Solute m < Buffer m Low Solute m = Buffer m Solute m > Buffer m High conductivity conductivity Low conductivity High conductivity Equivalent conductivity Low conductivity High conductivity Field strength Sample zone Sample zone Sample zone Peak shape
UV-VIS Detection Modes in CE Detection on-column decoupled high sensitivity cell
HP Capillaries for CE id [µm] l [cm] L [cm] Pathlength [µm] HP Extended Light Path capillaries 25 50 56 72 56 72 64.5 80.5 64.5 80.5 125 125 150 150 75 56 72 64.5 80.5 225 225 Standard capillaries 50 40 56 72 48.8 64.5 80.5 50 50 50 75 56 72 64.5 80.5 75 75 l = effective length from injection point to detector
Extended Light Path Capillaries Photograph of a dye front passing through the extended light path capillary detection region
HP Extended Light Path Capillary 1 50 µm 150 µm 2 3
HP Extended Light Path Capillary 60 40 mau Standard capillary id = 50 µm mau 60 40 Extended light path capillary id = 50, path 150 µm id = 50 µm l = 56 cm Inj. = 100 mbar s V = 25 kv i = 13 µa Buffer: 89 mm Tris-borate, ph 8.2 20 20 0 0 2 3 4 Time 5 6 [min] 2 3 4 Time 5 6 [min]
High Sensitivity Cell order of magnitude increase in sensitivity (S/N increase 10-fold) linear range up to 2200mAU (10^4) with 3% deviation from linearity enhanced spectral acquisition impurity determination <0.05% area/area decoupled design allows multiple re-use by capillary replacement
Detection Cell Design Fused silica body Flanking windows Removable capillary Black fused silica cell light path black fused silica cell reduces stray light light path extended to 1.2mm cell volume of 12nl flat windows flanking the cell improves spectral analysis Removable capillary
Capillary Modification 75µm i.d. capillaries are flared to 100µm i.d. and the outer diameter is beveled
Linear Range mau 2400 linear extension 2000 1600 1200 800 400 best fit 1% deviation on-column detection decoupled XXCell 1% deviation from linearity up to 1400mAU 3% deviation from linearity up to 2200mAU 0 10 20 30 40 50 60 70 80 90 100 % Tracer (2mM thiourea in water)
High Sensitivity Peptide Analysis: XxCell versus 75 µm Standard Capillary mau 40 30 20 XXCell w 75 mm id capillary capillary: 72cm eff x 75µm i.d. sample: 5pmol / µl tryptic digest of Myoglobin buffer: 50mM phosphate ph 2.5 detection: 200, 8 nm injection: 150mbar*s voltage: 20kV temp.: 25 C 10 0 75 mm id standard capillary 10 12 14 16 18 20 min
Impurity Determination: Ranitidine mau 40 35 impurities reported as % area/area of main peak 30 25 20 15 10 0.09 % Capillary: Fused Silica capillary 64cm (56cm eff) Buffer: 20mM borate ph 9.3 Injection: 200mbar*s Voltage: 30kV Temp.: 20 C Detection: HSDC, 225nm / 20nm 5 0 0.036 % -5 0 1 2 3 4 5 6 7 8 9 min
Summary of CE/CEC Presentation High efficiency and resolution Rapid separations Numerous modes to vary selectivity Wide application range Simple method development Automated instrumentation CEC has great "potential"