Basics of Capillary Electrophoresis. Ronald E. Majors Hewlett-Packard Wilmington, DE

Transcription

1 Basics of Capillary Electrophoresis Ronald E. Majors Hewlett-Packard Wilmington, DE

2 Outline Characteristics of CE EOF Phenomena Modes of Operation Zone Broadening Mechanisms Detection Modes

3 Characteristics of CE (I) Electrophoresis is performed in narrow-bore ( 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

4 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

5 Principle of CE Detector Inlet reservoir Exit reservoir Total length Effective length

6 Methods of Sample Injection Pressure Hydrodynamic Electrokinetic Sample Sample Vacuum Siphoning Sample Sample

7 Instrumental Aspects of CE Capillary thermostatting Diode-array detector Capillary cartridge HV Schematic of CE instrumentation Vial carousel (thermostatted) Buffer replenishment

8 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

9 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

10 Effect of Flow Profile on Zone Width EOF Laminar flow

11 µ EOF 4 3 Effect of ph on EOF in Various Capillary Materials 10 cm [ V s ] Pyrex Silica 2 1 Teflon ph

12 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

13 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

14 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

15 Capillary Zone Electrophoresis (CZE): Migration Superimposed on EOF in Fused Silica EOF

16 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 Neutral Anion (38.4 s) (50.7 s) Cation: Neutral: µ a ll Vt = = = µ EOF = = µ = µ µ =.. =. e a EOF (Note: µ will be negative for the anion) e v= ion velocity; u = electoosmotic velocity (cm /V 2 ) ; E= applied electric field (V/cm);

17 Capillary Zone Electrophoresis (CZE) in neutral coated capillary

18 CZE-Separations in PVA Capillaries ph3 mau ph Procainamide Tocainide Diisopyramide Pepsin inhomogen Pepsin Trypsin Inhibitor α-lactalbumin Myoglobin Carbonic Anhydrase II min 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

19 Micellar Electrokinetic Chromatography (MEKC) EOF

20 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

21 Volts MEKC of a Forensic Drug Screen b c d e j a f h g m n r i k q l p o 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

22 Capillary Gel Electrophoresis (CGE) - Separation based on Size replaceable polymer PVA / CEP coated capillary t < 0 t = 0 t > 0 sieving matrix

23 mau 40 Oligonucleotide Separation w/out Organic Addititve in Polymer Solution pd(a) without organic additive with organic additive 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

24 Separation of DNA Fragments mau bp 1605 bp bp 51 bp 65 bp 75 bp 396 bp 350 bp bp 179 bp bp 1198 bp 676 bp 460 bp 517 bp min 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: kvtemperature: 25 C; Detection: 260 nm with DAD filter for 260 nm (optional)

25 Isoelectric Focusing (IEF) IEF Coated Capillary filled with mixture of sample and ampholytes t = 0 Low ph ph gradient High ph t > 0

26 Capillary Isoelectric Focusing (CIEF) ph = 3 ph = 9 pi

27 CIEF - Hydrodynamic Mobilisation Comparison of Meat Extracts mau Beef Horse 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 min min

28 Isotachophoresis ITP T L t = 0 T L t > 0

29 ITP of Phosphorylated Nucleotides mau E-7 M Nucleotides in water Injection 5000 mbar s Leading Electrolyte Cl E-7 M Nucleotides in water + 35 mm NaCl min Trailing Electrolyte MES min

30 Origin of Electro - Osmotic Flow in CEC u eo = σ ε ε 0 r RT 2 2 cf η 1 / 2 E

31 Capillary ElectroChromatography (CEC): Principles and Practice

32 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

33 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

34 Principles of Electrochromatography Simplified system for CEC; 1 and 3 buffer vials, 2, FS capillary, µm i.d mm length, packed with HPLC packing material, 4 and 6, electrodes, 5, power supply, 7, point of detection.

35 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

36 Typical Example of CEC-separation mau 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 Thiourea Biphenyl Fluorene Fluoranthene Plate Number 65,000-80,000 Symmetry min

37 Achievable Efficiencies in µ-hplc and CEC Plates 250, , , ,000 50,000 0 µ-hplc 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 Capillary length [cm] 250, , , ,000 50,000 0 CEC 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

38 CEC Separations on Different Types of RP Packings 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 Diethylphthalate Biphenyl o-terphenyl isomeric esters of Diioctylphthalate min

39 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 Am dino2toluene 4. TNB NO2toluene 5. NB NO2toluene 6. TNT AMdiNO2toluene 7. 2,4-dinitrotoluene NO2toluene

40 Conclusions With CEC, isocratic, reversed phase, µhplc becomes feasible without significant instrumental constraints CEC overcomes high back-pressure difficulties associated with micro- LC with 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

41 Mechanisms of Band Broadening in CE Longitudinal diffusion Joule heating Solute-Wall interactions Electrodispersion

42 Joule Heating and Capillary Thermostatting Current [µa] 300 Ohm's Plot at 25 ºC 50 mm id caillary Liquid thermostating 10 m/s air thermostating No thermostating Voltage [kv]

43 Suppression of Solute - Wall Interactions Suppression dynamic coating - buffer ions - watersoluble polymers - surfactants permanent coating - chemically linked polymers - physically adhered polymers

44 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 Time [min]

45 Separation on permanently coated PVA Capillary mau untreated fused silica mau 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 min

46 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

47 UV-VIS Detection Modes in CE Detection on-column decoupled high sensitivity cell

48 HP Capillaries for CE id [µm] l [cm] L [cm] Pathlength [µm] HP Extended Light Path capillaries Standard capillaries l = effective length from injection point to detector

49 Extended Light Path Capillaries Photograph of a dye front passing through the extended light path capillary detection region

50 HP Extended Light Path Capillary 1 50 µm 150 µm 2 3

51 HP Extended Light Path Capillary mau Standard capillary id = 50 µm mau 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 Time 5 6 [min] Time 5 6 [min]

52 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

53 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

54 Capillary Modification 75µm i.d. capillaries are flared to 100µm i.d. and the outer diameter is beveled

55 Linear Range mau 2400 linear extension best fit 1% deviation on-column detection decoupled XXCell 1% deviation from linearity up to 1400mAU 3% deviation from linearity up to 2200mAU % Tracer (2mM thiourea in water)

56 High Sensitivity Peptide Analysis: XxCell versus 75 µm Standard Capillary mau 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 mm id standard capillary min

57 Impurity Determination: Ranitidine mau impurities reported as % area/area of main peak % 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 % min

58 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"